Elucidating the protein interactome of the
human ion channel TRPV4
A mutli-pronged approach to reveal the secrets of the fascinating
N-terminal TRPV4 protein interactome in vitro and in cellulo
Dissertation zur Erlangung des Grades
"Doktor der Naturwissenschaften" im Promotionsfach Chemie
am Fachbereich 09 - Chemie, Pharmazie, Geographie
und Geowissenschaften
der Johannes Gutenberg-Universität Mainz
Erika Diehl
née Pfeifer in Krasnoje, Republic of Moldova
May 2021

Dekan: 
1. Gutachterin: 
2. Gutachterin:
Tag des öffentlichen Promotionskolloquiums: 19.08.2021
D77 (Dissertation Universität Mainz)
3
Urheberschaftserklärung
Ich, Erika Diehl (née Pfeifer), versichere, dass ich die vorliegende Dissertation mit dem Titel
„Elucidating the protein interactome of the human ion channel TRPV4“ selbst verfasst, nicht
andere als die in ihr angegebenen Quellen oder Hilfsmittel benutzt, alle vollständig oder sin-
ngemäß übernommenen Zitate als solche gekennzeichnet sowie die Dissertation in der vor-
liegenden oder einer ähnlichen Form noch bei keiner anderen in- oder ausländischen Hochschule
anlässlich eines Promotionsgesuchs oder zu anderen Prüfungszwecken eingereicht habe.
4
Für meine Eltern, Familie und Freunde. Danke für Alles.
"Nature always wins but that doesn’t mean we have to bow or grovel to it. That maybe
even if we’re not always glad to be here, it’s our task to immerse ourselves anyway:
wade straight through it, right through the cesspool, while keeping eyes and hearts
open."
- The Goldfinch, Donna Tartt
5
Abbreviations
4α-PDD 4α-phorbol-12,13-didecanoate 5’,6’-EET 5’,6’-epoxyeicosatrienoic acid
A
AA arachidonic acid ACN acteonitrile
Abs absorbency AIP4 atropin-1-interacting protein 4 (ITCH)
ATP adenosine triphosphate ALS amyotrophic lateral sclerosis
AT1aR angiotensin receptor subtype 1a Avi avidin tag
ABC ATP-binding casette
B
BSA bovine serum albumin BmrA Bacilus subtilis multidrug resistance
ABC transporter ATP-binding Protein
C
CaM calmodulin CD circular dichroism
cDNA complementary DNA CFP cyan fluorescent protein
CTD C-terminal domain CV column volume(s)
CMT2C Charcot-Marie-Tooth Type 2C
D
ddH2O double distilled water DDX3X DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3
DNA desoxyribonulceic acid DSS disuccinimidyl suberate
DTT dithiothreitol DAG diacyl glycerol
DMF dimethylformamide
E
E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid
EM electron microscopy ER endoplasmatic reticulum
F
FA fetal akinesia F-BAR Fes/CIP4 homology Bin-Amphiphysin-Rvs
FDAB Familialr digital arthropathy-brachydaxtyly FRET Förster resonance energy transfer
G
GO gene ontology GPCR G-protein coupled receptor
GFP green fluorescence protein ggV4N Gallus gallus TRPV4 N-terminus
H
HEK293 cells human embrionic kidney 293 cells HECT homologous to E6-AP C-terminus
His6-tag hexahistidine-tag hsV4N human TRPV4 N-terminus
hsV4N Avi avidin-tagged human TRPV4 N-terminus hsV4 ARD human TRPV4 Ankyrin Repeat Domain
HSQC heteronuclear single quantum coherence
I
IEX ion exchange chromatography IMAC immobilized metal ion chromatography
IMS ion mobility spectrometry IP3 inositol-1,4,5-triphosphate
IPTG isopropyl β-D-1-thiogalactopyranosid ITCH E3 ubiquitin-protein ligase Itchy homolog
6
IDR intrinsically disordered region ITC isothermal calorimetry
M
MRW mean residue weight MW molecular weight
MS mass spectrometry / spectrometer MD metatropic dyplasia
N
NH amide group Ni2+-NTA Divalent nickel-nitrilotriacetic complex
NMR nuclear magnetic resonance NTD N-terminal domain
O
OD600 optical density at 600 nm
P
PACSIN protein kinase C and PBD phosphoinositide binding domain
casein kinase substrate in neurons
PCR polymerase chain reaction PDB protein data bank
PI(4,5)P2 Phosphatidylinositol-4,5-bisphosphate PLC Phospholipase C
PKA Protein kinase A PM Plasma membrane
PRR Proline rich region PPI protein-protein interaction
PTM posttranslational modification
R
RNA ribonucleic acid dxRNA duplex ribonucleic acid
RhoA Ras homolog family member A RBP RNA-binding protein
RT room temperature RNP granule ribonucleoprotein granule
S
SDS-PAGE sodium dodecyl sulfate- SH3 Src homology domain 3
polyacrylamide gel electrophoresis PCC Pearson correlation coefficient
SEC size exclusion chromatography SD standard deviation
Strep-POD streptavidin-peroxidase SG stress granule
T
TMD transmembrane domain Tris Tris(hydroxymethyl)aminomethane
TRP transient receptor potential Tx100 TritonX-100
TOF Time-of-flight
U
UDMSE ultra high-definition mass spectrometryE
W
wt wild type/ wild typic
X
XL-MS cross-linking mass spectrometry
Y
Y2H yeast-two hybrid
7
Summary
Transient receptor potential vanilloid 4 (TRPV4) is an integral membrane protein which func-
tions as a non-selective cation channel. TRPV4 plays a central role in mammalian sensory
perceptions like the sense of touch and temperature sensing. Although research steadily in-
creases, our knowledge of TRPV4 as well as other TRP superfamily members and their com-
plex regulation mechanisms still remain poorly understood. TRPV4 is also involved in not well-
characterized diseases, including peripheral neuropathies and skeletal dysplasias caused by
channel point mutations. Many of these point mutations are located in the human TRPV4 N-
terminus (hsV4N). The either solely skeletal or neuronal impact of these mutations led to the
assumption that hsV4N may interact with tissue specific proteins and that these protein-protein
interactions would then be specifically disturbed in the presence of disease-causing point mu-
tations. To this point, data on the protein interactome of hsV4N are fragmentary and further
investigations to shed light on the complex interaction network of TRPV4 are required.
In this work, recombinant hsV4N as well as the TRPV4 ankyrin repeat domain (hsV4 ARD),
as a part of hsV4N and notorious hot-spot for disease causing mutations, were investigated
with regard to their protein interactome in HEK293 cells using ultra high-definition mass spec-
trometry (UDMSE) in cooperation with the RG (University Medicine Mainz, JGU Mainz).
The interactome results revealed ribonucleotide binding proteins as a new potential class of
TRPV4 interacting proteins and indicate hsV4N as an until now unkown hub in the regulatory
mechanism of cytoplasmic ribonucleoprotein granule formation. These findings were under-
lined by the here shown direct interaction in vitro between recombinant hsV4 ARD and the
granule nucleating protein DDX3X. Interestingly, this interaction increased in the presence of
the neuropathy-causing R232C mutation in hsV4 ARD.
Another hsV4 ARD interacting protein studied in the course of this thesis is the small GTPase
RhoA. It was shown via nuclear magnetic resonance spectroscopy (NMR) that recombinantly
expressed 15N-RhoA directly interacts with hsV4 ARD and that this interaction significantly
decreases in the presence of the neuropathy-causing mutation R269C in hsV4 ARD (hsV4
8
ARD R269C). In a cooperation with the RG (Department of Neurology, Johns Hopkins
University School of Medicine) it was shown that this neuropathy mutation-dependent loss-of-
interaction leads to aberrant neurite-growth in cellulo and Drosophila melanogaster. These
findings strongly hint at that the disruption of TRPV4-RhoA interaction is one determinant of the
tissue-specific toxicity of TRPV4 neuropathy mutations.
An additional determinant for the tissue-specific toxicity of TRPV4 neuropathy mutations could
be the loss-of-interaction between neuropathy-causing TRPV4 R269C and the neurospecific
protein and known direct TRPV4-binding partner PACSIN1 that was demonstrated in this work.
PACSIN1 dampens hypotonicity-induced TRPV4-dependent Ca2+-influx in transiently trans-
fected HEK293 cells. This ability to desensitize TRPV4 to hypotonicity is lost in the presence of
the neuropathy-causing mutation TRPV4 R269C, probably due to a loss-of-interaction as shown
via co-immunoprecipitation. Strikingly, the PACSIN1-orthologue PACSIN3 retained its TRPV4
interaction and modulation upon hypotonicity. Further comprehensive studies with PACSIN
chimeras hint towards different binding mechanisms between TRPV4 and PACSIN1 or TRPV4
and PACSIN3, respectively.
Furthermore, in this thesis the foundation was laid to elucidate the possible role of post-translational
modifications in TRPV4s protein-protein interactions. It was shown that hsV4N directly interacts
with the E3 ubiquitin ligase ITCH and that lysine residues in close proximity to important regu-
latory sites within the intrinsically disordered region (IDR), preceding the hsV4 ARD in hsV4N,
are ubiquitinated.
Thus this thesis provides a comprehensive study of new potential TRPV4 protein interactors and
first explanations for the tissue-specificity of neuropathy-causing TRPV4 mutations.
9
Zusammenfassung
Transient receptor potential vanilloid 4 (TRPV4) ist ein integrales Membranprotein, das als nicht-
selektiver Kationenkanal fungiert. TRPV4 spielt eine zentrale Rolle bei Sinneswahrnehmungen
von Säugetieren wie dem Tastsinn und der Temperaturwahrnehmung. Obwohl die Forschung
über TRP-Kanäle stetig zunimmt, ist unser Wissen über TRPV4 sowie über andere Mitglieder
der TRP-Superfamilie und deren komplexe Regulationsmechanismen noch immer wenig ver-
standen. TRPV4 ist auch an nicht gut charakterisierten Krankheiten beteiligt, darunter periphere
Neuropathien und skeletale Dysplasien, die durch Punktmutationen im Kanal verursacht wer-
den. Viele dieser Punktmutationen befinden sich im N-Terminus von humanem TRPV4 (hsV4N).
Die entweder ausschließlich skeletale oder neuronale Auswirkung dieser Mutationen führte zu
der Annahme, dass hsV4N mit gewebespezifischen Proteinen interagieren könnte und dass
diese Protein-Protein-Interaktionen bei Vorhandensein von krankheitsverursachenden Punkt-
mutationen dann spezifisch gestört sein würden. Bis zu diesem Zeitpunkt sind die Daten über
das Proteininteraktom von hsV4N lückenhaft und weitere Untersuchungen, die mehr Klarheit
über das komplexe Interaktionsnetzwerk von TRPV4 bringen sollen, sind erforderlich.
In dieser Arbeit wurden rekombinantes hsV4N sowie die TRPV4-Ankyrin-Repeat-Domäne (hsV4-
ARD), als Teil von hsV4N und notorischer hot spot für krankheitsverursachende Mutationen,
hinsichtlich ihres Proteininteraktoms in HEK293-Zellen mittels Ultra-High-Definition-Massenspek-
trometrie (UDMSE) in Kooperation mit der RG (Universitätsmedizin Mainz, JGU Mainz)
untersucht. Die Interaktom-Ergebnisse enthüllten Ribonukleotid bindende Proteine als eine neue
potentielle Klasse von TRPV4-interagierenden Proteinen und weisen darauf hin, dass hsV4N
ein bisher unbekannter Knotenpunkt im Regulationsmechanismus von der Bildung von zyto-
plasmatischen Ribonukleoproteingranula sein könnte. Diese Erkenntnisse wurden durch die
hier gezeigte direkte Interaktion in vitro zwischen rekombinantem hsV4 ARD und dem Granu-
la nukleierenden Protein DDX3X untermauert. Interessanterweise verstärkte sich die Protein-
Protein-Interaktion von DDX3X mit hsV4 ARD in Gegenwart der neuropathieverursachenden
R232C-Mutation in hsV4 ARD.
10
Ein weiteres hsV4 ARD interagierendes Protein, das im Rahmen dieser Arbeit untersucht wur-
de, ist die kleine GTPase RhoA. Mittels Kernspinresonanzspektroskopie (NMR) wurde gezeigt,
dass rekombinant exprimiertes 15N-RhoA direkt mit hsV4 ARD interagiert und dass diese In-
teraktion in Gegenwart der Neuropathie verursachenden Mutation R269C in hsV4 ARD (hsV4
ARD R269C) signifikant abnimmt. In einer Kooperation mit der RG (Department of
Neurology, Johns Hopkins University School of Medicine) konnte gezeigt werden, dass die-
ser mutationsabhängige Interaktionsverlust zu aberrantem Neuriten-Wachstum in cellulo und
Drosophila melanogaster führt. Diese Befunde weisen stark darauf hin, dass die Störung der
TRPV4-RhoA-Interaktion eine Determinante für die gewebespezifische Toxizität von TRPV4-
Neuropathie-Mutationen ist.
Eine weiterer wichtiger Faktor für die gewebespezifische Toxizität von TRPV4-Neuropathiemuta-
tionen könnte der hier gezeigte Verlust der Interaktion zwischen der Neuropathie verursachen-
den TRPV4 R269C und dem neurospezifischen Protein und bekannten direkten TRPV4-Bin-
dungspartner PACSIN1 sein. PACSIN1 schwächt den Hypotonie induzierten TRPV4-abhängigen
Ca2+-Influx in transient transfizierten HEK293-Zellen ab. Diese Fähigkeit, TRPV4 gegenüber
Hypotonie zu desensibilisieren, geht in Gegenwart der Neuropathie verursachenden Mutation
TRPV4 R269C verloren Dies geschieht wahrscheinlich aufgrund eines Interaktionsverlustes,
wie durch Co-Immunopräzipitation gezeigt werden konnte. Auffallend ist, dass das PACSIN1-
Ortholog PACSIN3 seine TRPV4-Interaktion und -Modulation bei Hypotonie beibehielt. Weitere
umfangreiche Untersuchungen mit PACSIN-Chimären deuten auf unterschiedliche Bindungs-
mechanismen zwischen TRPV4 und PACSIN1 bzw. TRPV4 und PACSIN3 hin.
Weiterhin wurde in dieser Arbeit der Grundstein gelegt, um die mögliche Rolle von posttransla-
tionalen Modifikationen in den Protein-Protein-Interaktionen von TRPV4 aufzuklären. Es konnte
gezeigt werden, dass hsV4N direkt mit der E3-Ubiquitin-Ligase ITCH interagiert und dass Ly-
sinreste in unmittelbarer Nähe zu wichtigen regulatorischen Stellen innerhalb der intrinsisch
ungeordneten Region (IDR), die der hsV4-ARD in hsV4N vorgelagert ist, ubiquitiniert sind.
Somit liefert diese Arbeit eine umfassende Studie neuer potentieller TRPV4-Proteininteraktoren
und erste Erklärungen für die Gewebespezifität der Neuropathie verursachenden TRPV4-Muta-
tionen.
11
12
Contents
Contents
1 Introduction 17
1.1 A little grasp of the TRP channel world . . . . . . . . . . . . . . . . . . . . . . . 17
1.2 TRPV4 - the wild child amongst TRPV channels? . . . . . . . . . . . . . . . . . 21
1.3 Human TRP channels and their protein networks - glimpses of a brave new world 25
1.4 Aim of this thesis - taming the wild child TRPV4 . . . . . . . . . . . . . . . . . . 27
2 Materials 29
2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Buffer and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.4 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.5 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.6 Plasmids and expression constructs . . . . . . . . . . . . . . . . . . . . . . . . 46
2.7 Peptide and protein characteristics . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.8 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.9 Media and supplements for cell culture . . . . . . . . . . . . . . . . . . . . . . . 55
2.10 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.11 Laboratory equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.12 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3 Methods 59
3.1 General methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.1.1 Heat-shock transformation of E. coli bacteria . . . . . . . . . . . . . . . 59
3.1.2 Plasmid DNA preparation from E. coli . . . . . . . . . . . . . . . . . . . 59
3.1.3 Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . 59
3.1.4 Gibson Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.1.5 Agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . 61
13
Contents
3.1.6 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.1.7 SDS polyacrylamide gel electrophoresis (SDS-PAGE) . . . . . . . . . . 61
3.1.8 Western blot and chemiluminescence detection . . . . . . . . . . . . . . 62
3.1.9 Protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.1.10 Size exclusion chromatography . . . . . . . . . . . . . . . . . . . . . . . 64
3.2 Cell culture of eukaryotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.2.1 Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.2.2 Transient transfection of HEK293T cells . . . . . . . . . . . . . . . . . . 65
3.2.3 Stable transfection of HEK293 cells . . . . . . . . . . . . . . . . . . . . 66
3.3 Immunostaining and fluorescence microscopy . . . . . . . . . . . . . . . . . . . 67
3.4 Co-Immunoprecipitation in eukaryotic cells . . . . . . . . . . . . . . . . . . . . 68
3.5 Live Ca2+ imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.6 Ca2+ influx assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.7 Recombinant expression and purification of constructs . . . . . . . . . . . . . . 70
3.7.1 Recombinant expression of human N-terminal TRPV4 (hsV4N) constructs 70
3.7.2 Purification of hsV4N constructs . . . . . . . . . . . . . . . . . . . . . . 71
3.7.3 Purification of hsV4∆N122 and hsV4∆N132 constructs . . . . . . . . . 71
3.7.4 Biotinylation and purification of hsV4N constructs . . . . . . . . . . . . . 72
3.7.5 Recombinant expression and purification of human RhoA . . . . . . . . 72
3.7.6 Recombinant expression and purification of human DDX3X_aa122-582
(DDX3X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.7.7 Recombinant expression and purification of human ITCH . . . . . . . . . 74
3.7.8 Recombinant expression and purification of human ITCH WW domains . 75
3.7.9 Recombinant expression and purification of human YAP-WW domains . 75
3.8 Pulldown assay of biotinylated hsV4N constructs . . . . . . . . . . . . . . . . . 76
3.9 Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.9.1 In gel digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.9.2 In-solution digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.9.3 Liquid Chromatography Mass Spectrometry . . . . . . . . . . . . . . . . 78
3.9.4 Data Processing and Protein Identification . . . . . . . . . . . . . . . . . 79
3.10 Enzymatic assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.10.1 GTPase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.10.2 ATPase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.10.3 Ubiquitinylation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
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Contents
3.11 Blue Native-PAGE (BN-PAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
3.12 Circular dichroism (CD) spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 82
3.13 NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.14 Cross-linking mass spectrometry (XL-MS) . . . . . . . . . . . . . . . . . . . . . 83
4 Results 84
4.1 A beginning to elucidate the versatile TRPV4 N-terminal protein interactome - the
tip of the iceberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.1.1 Purification of biotinylated TRPV4 N-terminal proteins . . . . . . . . . . . 84
4.1.2 The TRPV4 N-terminal protein interactome in HEK293 cells derived via
mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.2 Birds of a feather flock together - the interaction between the two protean proteins
TRPV4 and DDX3X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.3 It is all about humanity - an in cellulo study of the interaction between human
TRPV4 and PACSIN1-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.4 Small but powerful - the small GTPase RhoA interacts with the TRPV4 Ankyrin
Repeat Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.5 All or nothing - interaction of ITCH requires the full TRPV4 N-terminus in vitro . . 126
4.6 TRPV4 and the actin cytoskeleton - connecting scientific disciplines . . . . . . . 133
5 Conclusion & Outlook 138
6 Appendix 143
6.1 Appendix - Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.2 Appendix - Purification of biotinylated TRPV4 N-terminal proteins . . . . . . . . 152
6.3 Appendix - A beginning to elucidate the versatile TRPV4 interatcome . . . . . . 152
6.4 Appendix - The interaction between the two protean proteins TRPV4 and DDX3X 177
6.5 Appendix - An in cellulo study of the interaction between human TRPV4 and
PACSIN1-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
6.6 Appendix -The small GTPase RhoA interacts with the TRPV4 Ankyrin Repeat
Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
6.7 Appendix - Interaction of ITCH requires the full TRPV4 N-terminus in vitro . . . . 184
6.8 Appendix - TRPV4 and the actin cytoskeleton - connecting scientific disciplines . 192
6.9 Appendix - Establishing HEK293 cell lines stably expressing TRP channels . . . 201
6.10 Appendix - Purification of ITCH WW domains . . . . . . . . . . . . . . . . . . . 202
15
Contents
6.11 Appendix - Purification of YAP1 WW domains . . . . . . . . . . . . . . . . . . . 203
6.12 Appendix - Amino acid and DNA sequences . . . . . . . . . . . . . . . . . . . . 204
7 List of Figures 225
8 List of Tables 227
9 Bibliography 230
16
Chapter 1. Introduction
1 Introduction
1.1 A little grasp of the TRP channel world
TRP stands for Transient Receptor Potential - a term introduced by Cosens et al. in a pub-
lication with the title "Abnormal Electroretinogram from a Drosophila Mutant".1 In this paper,
Cosens et al. described tran-
TRPC2
sient instead of steady electric (mouse) TRPM1 TRPM3 TRPM6
TRPC7
activities in Drosophila melano- TRPC3 TRPM7
TRPC6
gaster retinas upon light stim- TRPM4
TRPC5
uli, caused by a spontaneous
TRPM5
TRPC4
mutation in these flies. It took
another 20 years to identify TRPM2TRPC1
and characterize the respon-
TRPM8
TRPP2
sible Drosophila melanogaster
trp gene locus and to show TRPA1
TRPP1
that this gene encodes for
TRPV5
an integral membrane protein. TRPP3
In 1992 Baruch Minke and TRPV6
TRPML3
Roger Hardie showed this in- TRPV3
TRPML2 TRPV4
tegral membrane protein to be TRPML1 TRPV2 TRPV1
a Ca2+-gating ion channel.5 Figure 1.1: Multiple alignment phylogenetic tree of the mammalian TRP chan-
The identification of mam- nel superfamily. The phylogenetic tree shows the relations between the TRP
subfamilies based on sequence similarity. TRPP (polycystin), TRPC (canonical),
malian TRP channels not just
TRPM (melastatin), TRPA (ankyrin), TRPV (vanilloid) and TRPML (mucolipin). Note
only founded a whole new pro- that TRPC2 is a pseudogene in humans and therefore was substituted with the
mouse variant. Multiple sequence alignment and phylogenic tree generation was
tein superfamily, consisting of
performed with ClustalW2 at the EMBL-EBI server.2 Tree Rendering was performed
28 members divided into 6 sub- with DrawTree 3.66 via http://www.phylogeny.fr/.3,4
families (figure 1.1) - intensive
17
Chapter 1. Introduction
investigations also underpinned again a common catch in biochemical nomenclature: the initial
name often underestimates the plethora of physiological functions the proteins are covering -
and the TRP superfamily is a remarkable example for this.6
TRP channels are expressed at the plasma membrane and/or in intracellular organelle mem-
branes of almost every cell and tissue type. With their function as tetrameric cation channels
and slight preference for Ca2+, TRP channels play an important role in different physiological
processes including muscle contraction, cell proliferation or neurotransmitter release.6 In addi-
tion, since they couple intra- and extracellular signals to flux ions across the membrane, TRP
channels are also important mediators between the two membrane sides and are therefore well
suited to fulfill their role in somato-, osmo-, thermo- and photosensation as well as nocicep-
tion, amongst others sensory perceptions.7–13 In cellulo experiments revealed a broad diversity
among TRP channel activating/modulating stimuli, including ligand activation by exogenous
synthetic and natural chemical compounds or endogenous lipids and purine nucleotides as well
as temperature and cell swelling. This polymodality hints towards to a highly complex and
diverse regulation of TRP channels, defining their (patho)physiological role by the respective
cellular context in terms of the lipid- and proteome, for example. As diverse as the activa-
tion stimuli and physiological roles are the pathological outcomes of mutated TRP channels
- diseases which can be grouped into so called channelopathies.14–17 TRP channelopathies
span from kidney disorders (e.g. focal segmental glomerusclerosis, TRPC6 OMIM:603965,
polycystic kidney disease, TRPP2 OMIM:613095), skeletal dyplasias (e.g. metatropic dypla-
sia, TRPV4 OMIM:156530), lysosomal storage disorders (e.g. mucolipidosis type 4, TRPML1
OMIM:252650), skin disorders (e.g. erythrokeratodermia veriabilis et progressiva 6, TRPM4
OMIM: 618531) to neurodegenerative disorders (e.g. hereditary motor and sensory neuropathy
type IIC, TRPV4 OMIM:606071).
TRP channels share structural motifs among each other, but also exhibit structural diversity
- especially between the different TRP subfamilies. Each TRP monomer consists of struc-
turally conserved six transmembrane helices (S1-6) with a so-called reentrant loop between S5
and 6, forming the ion pore in a TRP tetramer (figure 1.2 A). S1-4 form the so-called voltage
sensor-like domain (VSLD), which affects channel gating by ligand binding.18–20 The biggest
structural differences between TRP channels are displayed within the intracellular N- and C-
termini as well as by large extracellular domains present in some subfamilies. Due to these
differences, the mammalian TRP superfamily can be divided into two groups. Group 1 (TRPM,
TRPA, TRPV and TRPC, figure 1.2 B) includes the TRP subfamilies with large cytosolic N-
termini,
18
Chapter 1. Introduction
Group 1
Reentrant loop extracellular
TRPV6 TRPA1
PDB:6d7s PDB:6pqq
S1-4 S5-6 S1-4 S5-6 membrane
intracellular
N C
Group 2
extracellular/
luminal
TRPML1 TRPP2 TRPC3 TRPM2
PDB:5wj5 PDB:5t4d PDB:6djr PDB:6mix membrane
intracellular
Figure 1.2: One representative structure of each human TRP subfamily. A Each TRP monomer consists of a structurally
conserved core of six transmembrane helices (S1-6) shown here as ribbon diagram (left) and topolgy model (right). B Selected
members belonging to group 1 of human TRP subfamilies, including TRPV6, TRPA1, TRPC3 and TRPM2. Monomers are high-
lighted in violet. For TRPV6, TRPA1 and TRPC3, only the ankyrin repeat domains (which are mostly α-helical structures) of the
whole cytosolic N-terminus of respective TRP channel are resolved. The preceding parts are putatively unstructured and therefore
not resolved in shown structures. For TRPM2 and the other TRPM channels a large so-called melastatin homology region defines
the N-terminus.21–24 C Selected members belonging to group 2 of human TRP subfamilies, including TRPML1 and TRPP2 with
their extracelluar/luminal domains spanning from the S1 to S2 transmembrane helix.
harboring pivotal regulatory sites like lipid binding sites or regulatory domains like ankyrin repeat
domains (ARDs). ARDs are α-helical motifs which serve as protein interaction platforms and
play a role in ion channel assembly.25–28 Group 2 TRP channels (TRPP amd TRPML, figure
1.2 C) are characteristic for their large domains spanning from S1 to S2, either being called
extracellular domain or extraluminal domain in the case of lysosomal localization of TRPML.
While structural investigations of (human) TRP channels immensely advanced in the past 10
years, the elucidation of human TRP channel protein interactomes is still underrepresented.20
Figure 1.3 shows current protein-protein interaction (PPI) networks deposited in the manually
curated TRP channel PPI database TRIP.29,30 Strikingly, most of the shown PPIs were deter-
mined with rodent proteins, whereas only a minority depicts PPIs with human proteins (see also
table 6.1). Besides the prominent Ca2+-binding protein Calmodulin and other calcium sensors
like ALG-2 and NCS-1, especially cytoskeletal associated (KIF3A, α-actinins, Tropomyosin 1)
and scaffolding proteins (AKAP5, NHERF-1, Homer-1) are represented. Furthermore, interac-
tors like Orai1, IP3R3 and RyR underline the role of TRP channels in store operated Ca2+
entry. Most of these proteins are part of large protein complexes to achieve physiological func-
19
Chapter 1. Introduction
tions and tightly regulate each other. As aberrant human TRP channels are the cause of sev-
eral, also tissue and cell-specific diseases, it is pivotal to determine these protein complexes
and ultimately also the regulation mechanisms between distinct PPIs to understand the patho-
physiological outcomes of mutated TRP channels. Here, the N- and C-termini of TRP channel
are especially interesting, as they
harbor important interaction sites
like ankyrin repeat domains but also TRPP
are accessible for a broad range
of possible protein interactors due
TRPC TRPM
to their cytosolic protrusion (figure
1.2 B). Almost all PPI studies with
TRP channels were conducted with
TRPA1
full-length channels. Additionally TRPML
to the need for a more compre-
TRPV
hensive knowledge of overall TRP
channel PPIs, it is also pivotal to
= human
determine the exact interaction sites = mouse/rat
at the channels. This leads to a
Figure 1.3: TRP channel PPI network extracted from the TRIP Database.
better understanding of these PPIs
The majority of the shown PPIs are with rodent proteins (grey nodes), whereas
and ultimately provide modulation only a minority depicts PPIs with human proteins (purple nodes). Node colors
of TRP subfamilies are indicated in the figure. See figure 6.1 for labeled nodes.
possibilities of aberrant PPIs due
Network was rendered via with Cytoscape 3.8.0.
to mutated TRP channels in dis- 29,30
eases.
20
Chapter 1. Introduction
1.2 TRPV4 - the wild child amongst TRPV channels?
In their review "The puzzle of TRPV4 channelopathies" published in 2013, Bernd Nilius and
Thomas Voets described TRPV4 as follows:
"TRPV4 could be compared to Proteus, a God of the sea in Greek mythology,
who has characteristics of flexibility, versatility, mutability and adaptability,
emerges in many shapes and can appear in frightening forms."31
This description is still surprisingly accurate, as in the course of this PhD thesis TRPV4 showed
another similarity to the sea god. Proteus is able to prophecy the future but is almost unwilling
to do so. Only to the ones who are able to capture him with cunning, Proteus will answer their
questions. And just as this moody sea god, TRPV4 is unwilling to easily reveal information
about its role in physiological and pathological processes. This PhD thesis shows that it took,
and it is still going to take, scientific highly versatile and multi-pronged approaches to "capture"
TRPV4 to get the desired information.
TRPV4 was first described as an osmoreceptor involved in systemic osmotic pressure in 2000
by Liedtke et al.32 Three years later, two research teams generated trpv4 null mice and pub-
lished the outcomes of this deletions in the murinal osmotic regulation.33,34 Liedtke and Fried-
mann determined systematic plasma hyperosmolality, diminished drinking and decreased va-
sopressin plasma levels in these mice. Vasopressin is a peptide hormone synthesized in the
hypothalamus and systematically released via the posterior pituitary in response to hyperos-
molality, inducing water reabsorbtion in the nephrons and arteriole constriction to increase the
peripheral vascular resistance and arterial blood pressure. Therefore, Liedtke and Friedmann
suggested that trpv4 null mice have an impaired osmosensation in the central nervous sys-
tem (CNS).33 In contrast, Mizuno et al. observed a normal drinking behavior in their trpv4
null mice and no plasma hyperosmolality. Furthermore, upon a hyperosmolal challenge due
to intraperitoneal propylene glycol injection, these mice showed increased vasopressin secre-
tion compared to wild type mice.34 Taken together, these studies and later conducted in cellulo
experiments show that TRPV4 is a pivotal key regulator in osmosensation - but how exactly
TRPV4 is involved in the vasopression-regulated osmolality homeostasis or even in the smaller
cellular context of osmolarity induced cell swelling or shrinkage still remains elusive. These
first descriptions of TRPV4 as a osmotically activated channel were then followed by the dis-
coveries of other TRPV4 activation and/or modulating factors such as innocuous temperature,
21
Chapter 1. Introduction
chemicals like the synthetic phorbol ester 4α-phorbol-12,13-didecanoate (4α-PDD) or the en-
dogenous compound 5’,6’-epoxyeicosatrienoic acid (5’,6’-EET) as well as plasma membrane
embedded phosphoinositides like phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2).35–39
Considering the relatively mild phenotypic consequences of trpv4 null mice, resulting in fer-
tile and viable mice with some impairments like the before mentioned altered osmosensa-
tion, impaired osteoclast differentation as well as hearing alteration and compromised vas-
cular endothelial functions, it is striking that mutations in TRPV4 lead to severe diseases in
humans.31,40–45 These autosomal-dominant hereditary TRPV4 channelopathies span from rel-
atively mild outcomes to heavily disabling or even lethal diseases which can be divided in two
main groups, namely skeletal dysplasias and peripheral neuropathies.14,15,31,46
Figure 1.4: Distribution of TRPV4 channelopa-
thy mutations. Shown residues of channelopathy-
causing TRPV4 mutations are mapped onto
the cryo-EM structure of X. tropicalis TRPV4
(PDB:6BBJ). Skeletal dysplasia mutants are
shown as green spheres, familial digital arthropa-
thy brachydactyly (FDAB) mutations as blue
spheres and peripheral neuropathy mutations as
yellow spheres. Whereas skeletal dysplasia mu-
tations are distributed over the whole channel,
neuropathy-causing mutations are mainly clustered
K276E in the cytosolic ankyrin repeat domain (hsV4 ARD).
R232C Numbered mutations correspond to the human
R269C TRPV4 sequence and were investigated in the
course of this thesis. The very N-terminal intrinsi-
cally disordered region (IDR, aa 1-149) is not re-
N solved and therefore not shown in the structure.
Skeletal dyplasias
Please note that a human TRPV4 structure, also
FDAB
Periph. neuropathies lacking the IDR, was published by Botte et al., but
no structure was deposited in the RSCB Protein
Data Bank to date.18
To date, five skeletal dysplasias and one other skeletal disease (familial digital arthropathy
brachydactyly, FDAB, OMIM: 606835) are known to be caused by TRPV4 mutations (OMIM:
605427). FDAB represents a very mild form of a TRPV4 mutation-induced bone disease, where
patients appear normal at birth but develop a deforming and painful osteoarthrithis due to sub-
chondral pathology of the finger- and toe phalanges as well as the metacarpal and -tarsal bones,
leading to arthropathy and brachydactyly. The FDAB-causing mutations in TRPV4 which are
known to date, were discovered in three unrelated families in the third ankyrin repeat (AR3)
22
Chapter 1. Introduction
of TRPV4 (G270V, R271P, F273L, see also blue spheres in figure 1.4).47 Unlike these FDAB-
associated mutations, skeletal dysplasia-causing mutations are not as tightly clustered in one
specific TRPV4 domain (figure 1.4). However, in sequential proximity, mutations (K276E or
I331F) lead to lethal metatropic dysplasia (MD), the severe end of TRPV4 channelopathies.48,49
Characteristic TRPV4-associated skeletal dysplasia phenotypes are brachydactyly, short trunk
and scoliosis.31 For lethal MD, additionally abnormally thick cartilage and congenital bone de-
formations were observed like hip dislocation and clubfeet, probably caused by altered endo-
chondral ossification during the fetal development.49 Recombinant overexpression - either in
HEK293T cell lines or Xenopus tropicalis oocytes - of skeletal dysplasia-inducing TRPV4 mu-
tations resulted mostly in gain-of-function channels, leading to increased Ca2+-levels in these
cells.50–52 The same observations in terms of elevated basal or activated Ca2+-influx activity
were also made with neuropathy-causing TRPV4 mutations.53,54 TRPV4-associated peripheral
neuropathies include two rare forms of spinal muscle arthropies (SMAs), namely congenital
distal SMAs (CDSMAs, OMIM:600-175) and scapuloperoneal SMA (SPSMA, OMIM:181405).
In most cases (approx. 90 %), the cause of SMA is a mutation in the survival of motorneu-
ron 1 (SMN1) protein, leading to ventral horn motor neuron degeneration and ultimately to
progressive muscle wasting of voluntary muscles in the limbs and also respiratory muscles.55
TRPV4-associated CDSMA and SPSMA primarily affect the muscles of lower limbs and for
SPSMA additionally the shoulder blade muscle. While TRPV4-associated CDSMA patients do
not have sensory deficits, SPSMA and the third TRPV4-associated neuropathy, Charcot-Marie-
Tooth Type 2C (CMT2C, OMIM:606071), share sensory deficits and vocal cord paresis as symp-
toms. Furthermore, CMT2C patients show, in contrast to the congenital conditions in CDSMA
and SPSMA, a progressive and non-congenital muscle weakness and atrophy of distal muscles.
Strikingly, mutations causing the before mentioned peripheral neuropathies especially cluster in
ankyrin repeats 2 and 4 of the TRPV4 ankyrin repeat domain, whereas skeletal dyplasia causing
mutations are spread all over the full-length channel, as shown in figure 1.4.27,31,49,56Amongst
the TRPV subfamily, only TRPV4 exhibits such a plethora of direct disease-causing mutations.
To date, only two other clinical syndromes are known to be caused by TRPV subfamily mem-
bers. TRPV3 is the until now only known cause of the Olmsted syndrome (mutilating palmo-
plantar keratoderma with periorificial keratotic plaques, OMIM: 614594), a very rare congenital
skin disorder leading to very thick skin on the hand palms and feet soles combined with severe
hyperkeratotic plaques around the mouth region. Olmsted syndrome-causing TRPV3 mutations
are either located in the loop between transmembrane helices S4 and 5 (G537S and G573C)
or in the C-terminus (W629G) and also result, like the disease-causing mutations in TRPV4, in
23
Chapter 1. Introduction
gain-of-function channels. On the other hand, most of the TRPV6 mutations leading to tran-
sient neonatal hyperparathyroidism (OMIM:618188) are loss-of-function mutations spread all
over the full-length channel.57 The impaired placental TRPV6 mediated Ca2+ influx induces
fetal hypocalcemia, leading to a secondary hyperparathyroidism where too much parathyroid
hormone (PTH) is secreted. The consequence is a metabolic bone disease in affected infants,
who present prenatal fractures, malformation of long bones and shortened ribs. In contrast
to other TRPV channel-associated diseases where treatment is exclusively symptomatic, post-
natal recovery was observed after oral Ca2+ supplementation.57 These findings underline the
importance of the TRPV ion flux fine tuning in respective tissues. TRPV channels can be di-
vided into two groups: thermosensitive and polymodal TRPVs (TRPV1-4), which show a low
basal ion channel activity and comparable low Ca2+ ion selectivity in comparison to the sec-
ond group, consisting of TRPV5 and 6. The latter two are Ca2+ selective with a constituent
activity and are not ligand-gated. It was shown that calmodulin (CaM) regulates TRPV5 and
6 in a negative, Ca2+-influx dependent feedback loop by blocking the cytosolic side of the ion
pore.58,59 Regarding the disease severity of TRPV4 gain-of-function mutations, TRPV4 seems
to be the wild child amongst the TRPV channels. The tissue-specificity of TRPV4-associated
diseases hints towards a vulnerability of these tissues due to the higher TRPV4-mediated Ca2+-
influx. Systemic antagonism of TRPV4 with the highly selective TRPV4 antagonist HC-067047
in mice lead to no severe side effects regarding parameters like motor coordination, fluid intake,
heart rate and thermoregulation.60 Additionally, the orally available, highly selective TRPV4 an-
tagonist GSK2798745 was recently examined in a phase I clinical trial in healthy subjects and
patients with cardiogenic pulmonary edema (NCT02119260). TRPV4 plays a central role in pul-
monary capillary endothelium, where the ion channel has a critical role in the development of
high pulmonary venous pressure (PVP)-associated lung edema after heart-failures. Pulmonary
capillary endothelial TRPV4 gets activated upon high PVP, leading to a reduced alveolar bar-
rier due to endothelial cell detachment in lungs. As a consequence, the pulmonary capillar-
ies show increased fluid (blood plasma) permeability and this leads ultimately to pulmonary
edema.61,62 GSK2798745 was well tolerated by healthy subjects and patients in terms of no
detectable changes in vital and clinical laboratory parameters. However, pulmonary edema
patients showed shortness of breath and reduced exercise tolerance upon GSK2798745 treat-
ment. Nevertheless, the patients did not show disease worsening. GSK2798745 laid there-
fore the foundation for a possible, future systemic TRPV4-antagonism in human diseases.61
As trpv4 null mice just show mild phenotypes as mentioned above and systemic, pharmaceu-
tically administrated TRPV4 antagonism seems to be well tolerated in mice and humans, it
24
Chapter 1. Introduction
is hypothesized that TRPV4-mediated Ca2+-influx can be compensated with other ion chan-
nels (or in the case of TRPV6-associated neonatal hyperparathyroidism even with nutritional
supplementation), but uncontrolled Ca2+-influx leads to pathophysiological consequences in
disease-related tissues. Several mechanisms were proposed, coupling TRPV4 mutations with
disease pathology, like calcium-induced cell death or altered neuritogenesis.31 But ultimately, all
these hypotheses aim towards either altered gene expression, probably through a Ca2+/CaM-
dependent pathway or altered protein-protein interactions between TRPV4 and cell-type spe-
cific protein complexes. Especially for the neuropathy-causing mutations, which mainly cluster
in the cytosolic TRPV4 N-terminus, altered tissue-specific PPIs could be the reason for the
neurospecificity of these mutations.
1.3 Human TRP channels and their protein networks - glimpses of
a brave new world
In the last decades, several high-throughput methods to identify protein interactomes emerged
and were optimized continuously, such as protein-fragment complementation assay screens
(PFCAs) like the yeast two-hybrid assay (Y2H) or mass spectrometry (MS) approaches. PF-
CAs map binary PPIs in the context of defined libraries of probable interactors, whereas MS
approaches can provide qualitative and quantitative information of PPIs in complex protein
networks.63,64 The characterization of ion channel interactomes with MS-based proteomics
greatly contributed to a better understanding of these supramolecular complexes, like for the
glutamate receptors of the AMPA-type (AMPARs).65–67 AMPARs are cation channels localized
in the central nervous system, where they are involved in signal transmission in glutamatergic
synpases. AMPARs are activated by the neurotransmitter glutamate, but also by the synthetic
glutamate analog AMPA.67 Based on classical molecular cloning, AMPARs were assumed to
consist of four GluA transmembrane proteins, which are surrounded by up to four auxiliary
subunits, which are either the so called transmembrane AMPAR regulatory proteins (TARPs),
chornichon homologs or the GSG1-l protein. However, comprehensive MS-based proteomics
revealed that AMPARs are more complex supramolecular constructs which assemble from a
pool of 34 proteins known to date. From these 34 proteins, 21 were newly identified via MS, un-
derlining the strength of unbiased and hypothesis-free MS-based proteomics.Such MS-based
approaches provide roadmaps for further in-depth and structural investigations of the molecular
25
Chapter 1. Introduction
framework for the highly complex cell physiology of not only AMPARs, but also other ion chan-
nels.
Surprisingly, human TRP channel PPIs listed in table 6.1 were either determined via fusion
protein-pull down assays with subsequent western blotting or classical Y2H assays. Only for
two human TRP channels, namely TRPM4 and TRPV4, MS-based human protein interactome
studies are published to date, both conducted in HEK293T cells. For human TRPM4, Caceres
et al. identified proteins with actin cytoskeleton-related functions as the largest group of putative
TRPM4 interactors, whereat McCray et al. identified a more diverse range of protein as putative
TRPV4 interactors, with the small GTPase RhoA as one of the most significant ones (see also
section 4.4 for a more detailed description of this PPI).68,69 Both approaches were performed
with transiently transfected HEK293T cells, expressing the respective full-length channel fused
with an affinity tag. This affinity tag enables the purification of the tagged (bait) protein from
a cell extract via affinity enrichment, co-purifying putative interactors which are then identified
by mass spectrometry.64 While this approach provides information about the putative interac-
tome of the full-length channels, it is not possible to determine the exact interaction sites of
the putative interactors within the channel. The other known (mostly rodent) TRPV4 interactors
were mainly determined with non MS-based approaches like Y2H or co-immunoprecipitations
(co-IPs) and can be mainly divided into three categories (for a list extracted from the TRIP
database see table 6.2). First category compromises cytoskeletal and cytoskeletal associ-
ated proteins like α-arrestin 1-2, tubulins, α-actin and PACSIN1-3, indicating TRPV4s role in
mechanosensitivity.70,71 The second category includes interactors which modify TRPV4 post-
translationally, like the tyrosin-kinases LYN and FYN or the E3 ubiquitin ligase ITCH. The third
category includes transmembrane proteins like other TRP channels or AQP-4, for example.
Most of the co-IPs were combined with site-directed mutagenesis to identify the exact TRPV4
interaction site, like for one of the most prominent TRPV4 interactors PACSIN3.70,71 Whereat
approaches like before mentioned co-IPs with site directed mutagenesis of probable interac-
tion sites are able to provide information about direct PPIs, the restriction to binary PPI studies
compromises the speed but also an comprehensive overview of putative supramolecular com-
plexes. Overall, little is known about the TRPV4 protein interactome. To get a comprehensive,
unbiased overview of a complex proteome or interactome, the highly sophisticated ultra high-
definition mass spectrometryE (UDMSE) - first presented by Distler et al. - is the method of
choice.72 Shortly described, UDMSE increases drastically the number of identified peptides in
a complex mixture by introducing collision-energy dependent traveling wave-based ion-mobility
separation (CE-dependent IMS) into liquid-chromatography coupled mass spectrometry (LC-
26
Chapter 1. Introduction
MS). With this, the precursor ion fragmentation efficiency improves significantly, enhancing the
analytical depth of unbiased data-independent data acquisition.72 With this and by additionally
developing a robust and comprehensive in-house software for UDMSE data analysis ,named
ISOQuant, Distler and Kuharev et al. increased the numbers of identifiable protein groups com-
pared to other generic MS set-ups significantly.73,74 With this, UDMSE is an excellent label-free
and quantitative method to elucidate the protein interactome of TRPV4, which is involved in
several severe channelopathies, as mentioned above. Due to the accumulation of disease-
causing mutations in the cytosolic TRPV4 N-terminus (hsV4N), this domain was chosen as a
starting point to elucidate the TRPV4 protein interactome in HEK293 cells. The high sensitivity
and in-depth protein coverage of UDMSE is essential for future TRPV4 interactome investiga-
tions in TRPV4 channelopathy-affected tissues, as this method is particularly suitable to identify
altered tissue-specific protein-protein interactions as a possible reason for the disease pathol-
ogy and tissue specificity of respective TRPV4 mutations.27 Therefore, UDMSE results serve
as an excellent guidance for further in-depth investigations of determined TRPV4 interactome
members.
1.4 Aim of this thesis - taming the wild child TRPV4
The TRPV4 N-terminus is a notorious hot-spot of disease-causing mutations, which mostly
result in gain-of-function ion channels and dysregulated Ca2+-influx.49,53,54 Interestingly, es-
pecially the N-terminus harbors a significant amount of mutation sites which cause peripheral
neuropathies like Charcot-Marie-Tooth Type 2C (CMT2C).54,56,75 As the TRPV4 N-terminus con-
sists of important protein interaction sites like the ankyrin repeat domain (ARD) or the proline
rich region (PRR) and due to its cytosolic protrusion, this thesis elucidates the PPIs of the
human (mutated) TRPV4 N-terminus (hsV4N) with a multipronged approach in vitro and in
cellulo. For this, a major focus of this thesis was to elucidate the overall HEK293 cell pro-
tein interactome of hsV4N via ultra high-definition mass spectrometryE (UDMSE) and to narrow
down the interaction sites by conducting identical UDMSE experiments with the isolated TRPV4
ARD (hsV4 ARD). After data evaluation and protein-protein network analysis via gene ontol-
ogy enrichment (GO), for example, promising interaction candidates were worked out. Possible
direct interactions between hsV4N and respective candidates were then investigated with a
versatile spectrum of methods, spanning from in cellulo experiments like live Ca2+-imaging, co-
immunoprecipitations and fluorescence microscopy to the very detailed elucidation of PPIs with
27
Chapter 1. Introduction
nuclear magnetic resonance spectroscopy and cross-linking mass spectrometry. Ultimately, this
thesis laid the foundation to understand the tissue specific disease outcomes of TRPV4 muta-
tions and for possible future regulations of the pathological Ca2+-influx, which is especially
interesting for the progressive motoneuron-degeneration in CMT2C.
28
Chapter 2. Materials
2 Materials
2.1 Chemicals
Table 2.1: Chemicals
Chemical Supplier
Acetic acid 100 % (AcOH) Roth, Karlsruhe
Acrylamide/Bisacrylamide 37.5 % Roth, Karlsruhe
Acetonitrile LC-MS grade (ACN) Roth, Karlsruhe
15N-Ammonium chloride (15NH4Cl) CIL Inc., Tewksbury (USA)
Ammonium peroxisulfate (APS) Roth, Karlsruhe
Adenosinediphosphate (ADP) Roth, Karlsruhe
Adenosinetriphosphate (ATP) Roth, Karlsruhe
Agar Agar Roth, Karlsruhe
Agarose Roth, Karlsruhe
Ampicillin sodium salt (Amp) Roth, Karlsruhe
L-Ascorbic Acid Sigma-Aldrich, Munich
D-Biotin Roth, Karlsruhe
Dynabeads™M-280 Streptavidin Invitrogen, Darmstadt
Dynabeads™Protein G Invitrogen, Darmstadt
Bovine serum albumin (BSA) Roth, Karlsruhe
Benzamidine hydrochlorid Sigma-Aldrich, Munich
Bromphenol blue sodium salt Roth, Karlsruhe
3-[(3-Cholamidopropyl)dimethylammonio]- Roth, Karlsruhe
1-propanesulfonate (CHAPS)
Coomassie Brilliant Blue G-250 Dye Roth, Karlsruhe
Coomassie Brilliant Blue R-250 Dye Roth, Karlsruhe
29
Chapter 2. Materials
Chemical Supplier
Calcium chloride (CaCl2) Roth, Karlsruhe
CHS (Cholesteryl hemisuccinate) Sigma Aldrich, Munich
Digitonin Roth, Karlsruhe
Dithiothreitol (DTT) Sigma-Aldrich, Munich
Dimethylsulfoxide (DMSO),
Headspace Grade or BioScience-Grade Roth, Karlsruhe
4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich, Munich
Ethanol 99.9 % p.a. (EtOH) Roth, Karlsruhe
Ethidium bromide 99.9 % AppliChem, Darmstadt
Formic acid Optima LC/MS (FA) Thermo Scientific, Waltham (USA)
Fura 2-AM Invitrogen, Darmstadt
Glycerol Roth, Karlsruhe
D-Glucose Roth, Karlsruhe
GSK-1016790A (GSK-101, TRPV4 agonist) Sigma-Aldrich, Munich
HC-067047 (HC-067, TRPV4 antagonist) Sigma-Aldrich, Munich
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Sigma-Aldrich, Munich
Hydrochloric acid (HCl) Roth, Karlsruhe
Isopropyl-β-D-thiogalactopyranoside (IPTG) Roth, Karlsruhe
Isopropyl alcohol Roth, Karlsruhe
Imidazol Roth, Karlsruhe
Kanamycin sulphate Roth, Karlsruhe
Lipofectamin LTX Invitrogen, Darmstadt
Methanol 99.9 % p.a. (MeOH) Roth, Karlsruhe
Magnesium chloride hexahydrate (MgCl2·6 H2O) Roth, Karlsruhe
Magnesium sulfate heptahydrate (MgSO4·7 H2O) Roth, Karslruhe
Mowiol® 4-88 Sigma-Aldrich, Munich
n-Dodecyl β-D-maltoside (DDM) Sigma-Aldrich, Munich
β-nicotinamide adenine dinucleotide (NADH) Sigma-Aldrich, Munich
Nickel nitrilotriacetic acid (Ni2+-NTA) agarose QIAGEN, Hilden
Paraformaldehyde (PFA) Sigma-Aldrich, Munich
Pierce™Lysis Buffer Invitrogen, Darmstadt
CST Protease/Phosphatase Inhibitor Cocktail (100X) CST, Danvers (USA)
30
Chapter 2. Materials
Chemical Supplier
Precision Plus Protein™ Dual Color Standards BioRad, Hercules (USA)
Phalloidin-TRITC Sigma-Aldrich, Munich
Potassium ethylenediaminetetraacetic acid (K-EDTA) Roth, Karlsruhe
Potassium hydroxide (KOH) Roth, Karslruhe
Phenylmethylsulfonylfluoride (PMSF) Sigma-Aldrich, Munich
Phosphoenolpyruvate (PEP) Sigma-Aldrich, Munich
Potassium chloride (KCl) Roth, Karlsruhe
PIPES (piperazine-N,N’-bis(2-ethanesulfonic acid)) Roth, Karlsruhe
Roti®-Lumin 1 and 2 Roth, Karlsruhe
SIGMAFAST Protease Inhibitor
Cocktail Tablet, EDTA-Free Sigma-Aldrich, Munich
Sodium chloride (NaCl) Roth, Karlsruhe
Sodiumdodecylsulfate (SDS) Roth, Karlsruhe
Sodium hydroxide (NaOH) Roth, Karlsruhe
Sucrose Roth, Karlsruhe
Tetramethylethylendiamine (TEMED) Roth, Karlsruhe
Tris(hydroxymethyl)aminomethane (Tris) Roth, Karlsruhe
Triton X-100 Roth, Karlsruhe
Tween® 20 Roth, Karlsruhe
Water, HPLC gradient grade Roth, Karlsruhe
Water, LC-MS grade Roth, Karlsruhe
2.2 Buffer and solutions
Table 2.2: General buffer and solutions
Buffer/solution Ingredients
10x Running buffer pH = 8.3 20 mM Tris
1.54 M Glycin
31
Chapter 2. Materials
Buffer/solution Ingredients
1x SDS-PAGE-Running buffer pH = 8.3 1x Running buffer
0.1 % (v/v) SDS
1x SDS-PAGE Wet blot buffer 1x Running buffer
20 % (v/v) EtOH
4x SDS-PAGE sample buffer 200 mM Tris/HCl pH = 6.8
400 mM DTT
8 % (v/v) SDS
0.4 % (w/v) Bromphenole blue
40 % (v/v) Glycerol
10x PBS pH = 7.4 2.7 mM KCl
10 mM Na2HPO4
2 mM KH2PO4
137 mM NaCl
1x PBS/T pH = 7.4 1x PBS
0.05 % (v/v) Tween® 20
2x HEPES pH = 7.5 12 mM Glucose
50 mM HEPES
10 mM KCl
280 mM NaCl
1.5 mM Na2HPO4· 2 H2O
Coomassie staining solution 1 % (w/v) Coomassie blue R250
40 % (v/v) MeOH
10 % (v/v) Acetic acid
Coomassie destaining solution 10 % (v/v) Acetic acid
LB-Medium 25 g/L LB-Medium (Luria/Miller Broth) in MP-H2O
TB-Medium 47.5 g/L TB-Medium (Terrific Broth) in MP-H2O
Stripping buffer 100 mM NaOH
2 % (w/v) SDS
0.5 % (w/v) DTT
Mobile Phase A 0.1 % (v/v) FA
3 % DMSO
Ultrapure Water
32
Chapter 2. Materials
Buffer/solution Ingredients
Mobile Phase B 300 mM NaCl
Ultrapure Water
M9 Medium 15 g/L KH2PO4
33.9 g/L Na2HPO4
0.75 g/L 15NH4Cl
2.5 g/L NaCl
4 g/L Glucose
2 mM MgSO4
10 µM FeCl3
Trace elements 10 mL/L
Magic Mix 1 mL/L
Trace Elements 0.2 mg/mL CaCl2·2H2O
0.2 mg/mL ZnSO4·7H2O
0.2 mg/mL MnSO4·H2O
5 mg/mL Thiamine
5 mg/mL Niacin
0.1 mg/mL Biotin
Magic Mix 1 Centrum Vitamin Tablet in 20 mL H2O
Artifical CSF (aCSF) buffer 8.185 g/L NaCl
0.360 g/L KCl
0.203 g/L MgCl2·6H2O
0.294 g/L CaCl2·2H2O
4.320 g/L D-Glucose
2.350 g/L HEPES pH = 7.4
Hypotonic Artifical CSF (aCSF) buffer 0.360 g/L KCl
0.203 g/L MgCl2·6H2O
0.294 g/L CaCl2·2H2O
4.320 g/L D-Glucose
2.350 g/L HEPES pH = 7.4
Pulldown lysis buffer 412.0 g/L Sucrose
12.0 g/L PIPES pH = 6.8
23.2 g/L NaCl
33
Chapter 2. Materials
Buffer/solution Ingredients
2.6 g/L MgCl2·6 H2O
187.5 mg/L Digitonin
Electrophoresis cathode buffer pH = 8.3 25 mM Tris
192 mM Glycine
0.02 % Coomassie-G250
Table 2.3: Buffer and solutions for purification of hsV4N, hsV4N∆122 and hsV4N∆132 con-
structs. hsV4N∆132 is also referred to as hsV4 ARD, hsV4N∆122 as hsV4 ARD-PRR.
Buffer/solution Ingredients
E.coli lysis buffer hsV4N and hsV4N∆132 20 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
0.1 % (v/v) Triton X-100
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
Wash 1 20 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
Wash 2 50 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
Elution 500 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
SEC buffer 10 mM Tris-HCl pH = 7
1 mM K-EDTA pH = 7
34
Chapter 2. Materials
Buffer/solution Ingredients
300 mM NaCl
1 mM DTT
1 mM Benzamidine
Table 2.4: Buffer and solutions for purification of hsRhoA
Buffer/solution Ingredients
E.coli lysis buffer RhoA 200 mM NaCl
50 mM HEPES pH = 7.5
0.1 % (v/v) Triton X-100
10 % (v/v) Glycerol
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash 1 50 mM Imidazol pH = 7.5
200 mM NaCl
50 mM HEPES pH = 7.5
5 % (v/v) Glycerol
IMAC Elution 500 mM Imidazol pH = 7.5
200 mM NaCl
50 mM HEPES pH = 7.5
10 % (v/v) Glycerol
TEV dialysis buffer 200 mM NaCl
25 mM Tris-HCl pH = 7
1 mM DTT
0.5 mM K-EDTA
TEV: RhoA 1 to 30 molar ratio
Reverse IMAC buffer 10 mM Imidazol pH = 7.5
35
Chapter 2. Materials
Buffer/solution Ingredients
200 mM NaCl
50 mM HEPES pH = 7.5
SEC buffer 200 mM NaCl
50 mM HEPES pH = 7.5
5 mM MgCl2
Table 2.5: Buffer and solutions for purification of hsDDX3X_aa122-582 (DDX3X)
Buffer/solution Ingredients
E.coli lysis buffer hsDDX3X 10 mM Imidazol pH = 7.4
400 mM NaCl
50 mM NaH2PO4 pH = 7.4
0.1 % (v/v) Triton X-100
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash A buffer 10 mM Imidazol pH = 7.4
400 mM NaCl
50 mM NaH2PO4 pH = 7.4
IMAC Wash B buffer 20 mM Imidazol pH = 7.4
400 mM NaCl
50 mM NaH2PO4 pH = 7.4
IMAC High Salt buffer 1 M NaCl
50 mM NaH2PO4 pH = 7.4
IMAC Elution buffer 300 mM Imidazol pH = 7.4
150 mM NaCl
50 mM NaH2PO4 pH = 7.4
TEV dialysis buffer 300 mM NaCl
36
Chapter 2. Materials
Buffer/solution Ingredients
25 mM NaH2PO4 pH = 7
1 mM DTT
TEV: DDX3X 1 : 30 molar ratio
Reverse IMAC buffer 10 mM Imidazol pH = 7.4
300 mM NaCl
25 mM NaH2PO4 pH = 7.4
SEC buffer 125 mM NaCl
25 mM HEPES pH = 7.4
Table 2.6: Buffer and solutions for purification of hsITCH
Buffer/solution Ingredients
E.coli lysis buffer hsITCH 200 mM NaCl
50 mM Tris-HCl pH = 8
0.1 % (v/v) Triton X-100
5 % (v/v) Glycerol
0.1 % (v/v) DDM
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash 1 25 mM Imidazol pH = 8
200 mM NaCl
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
IMAC Elution 500 mM Imidazol pH = 8
200 mM NaCl
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
37
Chapter 2. Materials
Buffer/solution Ingredients
TEV dialysis buffer 200 mM NaCl
50 mM Tris-HCl pH = 8
1 mM DTT
TEV: ITCH 1 : 30 molar ratio
Reverse IMAC buffer 10 mM Imidazol pH = 8
200 mM NaCl
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
SEC buffer 200 mM NaCl
50 mM Tris-HCl pH = 8
1.5 % (v/v) Glycerol
Table 2.7: Buffer and solutions for purification of hsITCH WW domain constructs
Buffer/solution Ingredients
E.coli lysis buffer hsITCH WW domains 200 mM NaCl
50 mM Tris-HCl pH = 8
0.1 % (v/v) Triton X-100
5 % (v/v) Glycerol
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash 1 25 mM Imidazol pH = 8
200 mM NaCl
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
IMAC Elution 500 mM Imidazol pH = 8
200 mM NaCl
38
Chapter 2. Materials
Buffer/solution Ingredients
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
TEV dialysis buffer 200 mM NaCl
50 mM Tris-HCl pH = 8
1 mM DTT
TEV: ITCH 1 : 30 molar ratio
Reverse IMAC 10 mM Imidazol pH = 8
200 mM NaCl
50 mM Tris-HCl pH = 8
5 % (v/v) Glycerol
SEC buffer 200 mM NaCl
50 mM Tris-HCl pH = 8
Table 2.8: Buffer and solutions for purification of TRPC 3, 4 and 6 ARDs
Buffer/solution Ingredients
E.coli lysis buffer TRPC ARDs 20 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
0.1 % (v/v) Triton X-100
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash 1 20 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
Wash 2 50 mM Imidazol pH = 8
300 mM NaCl
39
Chapter 2. Materials
Buffer/solution Ingredients
20 mM Tris-HCl pH = 8
Elution 500 mM Imidazol pH = 8
300 mM NaCl
20 mM Tris-HCl pH = 8
SEC buffer 10 mM Tris-HCl pH = 7
1 mM K-EDTA pH = 7
300 mM NaCl
1 mM DTT
1 mM Benzamidine
Table 2.9: Buffer and solutions for purification of hsYAP-WW domains
Buffer/solution Ingredients
E.coli lysis buffer hsYAP WW domains 20 mM Imidazol pH = 7.4
120 mM NaCl
30 mM HEPES pH = 7.4
0.1 % (v/v) Triton X-100
add fresh:
1 mM Benzamidine
1 mM PMSF
1 SIGMAFAST Protease Inhibitor
RNAse, DNAse, Lysozyme
IMAC Wash 1 20 mM Imidazol pH = 8
120 mM NaCl
30 mM HEPES pH = 8
IMAC Wash 2 50 mM Imidazol pH = 8
120 mM NaCl
30 mM HEPES pH = 8
IMAC Wash 3 75 mM Imidazol pH = 8
120 mM NaCl
40
Chapter 2. Materials
Buffer/solution Ingredients
30 mM HEPES pH = 8
IMAC Elution 500 mM Imidazol pH = 8
120 mM NaCl
30 mM HEPES pH = 8
TEV dialysis buffer 300 mM NaCl
25 mM Tris-HCl pH = 7
1 mM DTT
TEV: hsYAP-WW domains 1 : 30 molar ratio
Reverse IMAC buffer 10 mM Imidazol pH = 8
120 mM NaCl
30 mM HEPES pH = 8
SEC buffer 120 mM NaCl
30 mM HEPES pH = 7.4
2.3 Enzymes
Table 2.10: Enzymes
Enzymes Supplier
Desoxyribonuclease I (DNAse) Sigma-Aldrich, Munich
DpnI NewEngland BioLabs, Frankfurt a.M.
HiFi Polymerase Kapa Biosystems, Wilmington (USA)
Lactate Dehydrogenase (LDH) Sigma-Aldrich, Munich
Lysozyme Sigma-Aldrich, Munich
Ribonuclease A (RNAse) Sigma-Aldrich, Munich
T5 Exonuclease NewEngland BioLabs, Frankfurt a.M.
Phusion DNA Polymerase NewEngland BioLabs, Frankfurt a.M.
Pyruvate Kinase (PK) Sigma-Aldrich, Munich
Taq DNA Ligase NewEngland BioLabs, Frankfurt a.M.
41
Chapter 2. Materials
Enzymes Supplier
E1 Ubiquitin ligase UBE1 Prof. Sumner, Baltimore (USA)
E2 Ubiquitin ligase Ubc5Hc Prof. Sumner, Baltimore (USA)
2.4 Antibodies
Table 2.11: Antibodies
Antibody Supplier
Anti-ANXA2 mouse Sigma-Aldrich, Munich
Anti-DDX3X rabbit Sigma Aldrich, Munich
Anti-GFP mouse (3E6) Invitrogen, Darmstadt
Anti-GFP mouse Sigma-Aldrich, Munich
Anti-FLAG mouse CST, Danvers (USA)
Anti-FLAG rabbit CST, Danvers (USA)
Anti-V5 mouse CST, Danvers (USA)
Anti-myc mouse CST, Danvers (USA)
Anti-His6-HRP Invitrogen, Darmstadt
Streptavidin-POD (Strep-POD) Roche, Basel (CH)
Horse anti-mouse HRP-linked IgG CST, Danvers (USA)
Goat anti-rabbit HRP-linked IgG CST, Danvers (USA)
Alexa-Fluor-488 goat anti-mouse Invitrogen, Darmstadt
Alexa-Fluor-555 goat anti-rabbit Invitrogen, Darmstadt
Alexa Fluor-647 goat anti-mouse Invitrogen, Darmstadt
Alexa Fluor-647 goat anti-rabbit Invitrogen, Darmstadt
42
Chapter 2. Materials
2.5 Oligonucleotides
The oligonucleotides in tables 2.12 and 2.13 were designed in the course of this work and were
obtained from Sigma-Aldrich (Munich). For the estimation of the melting temperatures equation
2.1 was used. The nomenclature of the primers refers to the employed nomenclature at the
Hellmich workgroup.
Tm = 4 · #G and C + 2 · #A and T (2.1)
Table 2.12: Oligonucleotides for Quickchange PCR
′ ′
Designation Sequence (5 –3 ) Length Tm [°C]
human TRPV4 S134A_for GAGAAGCAGCCGCAGGAACCCAAAGCC 27mer 71
human TRPV4 S134A_rev GTCGGCGTCCTTGGGTTTCGGGGAC 25mer 70
human TRPV4 S134E_for GAGAAGCAGCCGCAGGAACCCAAAGAA 27mer 67
human TRPV4 S134E_rev GTCGAAGTCCTTGGGTTTCGGGGAC 25mer 65
human TRPV4 E183K_for CGCCTAACTGATAAGGAGTTTCGAG 25mer 70
human TRPV4 E183K_rev GGCTCTCGAAACTCCTTATCAGTTAG 26mer 70
human TRPV4 L199F_for GCCCAAGGCCTTTCTGAACCTGAGC 25mer 75
human TRPV4 L199F_rev CATTGCTCAGGTTCAGAAAGGCCTTG 26mer 71
human TRPV4 R232C_for CATTAACTCGCCCTTCTGTGACATC 25mer 70
human TRPV4 R232C_rev GATAGTAGATGTCACAGAAGGGCGAG 26mer 72
human TRPV4 R269C_for GCCCAGGCCTGTGGGCGCTTCTTC 24mer 78
human TRPV4 R269C_rev GCTGGAAGAAGCGCCCACAGGCCTG 25mer 78
human TRPV4 R271P_rev CCCGTGGGCCCTTCTTCCAGCC 22mer 75
human TRPV4 R271P_rev CTTGGGCTGGAAGAAGGGCCCACG 24mer 76
human TRPV4 K276E_for CGCTTCTTCCAGCCCGAGGATGAG 24mer 75
human TRPV4 K276E_rev CCCCCTCATCCTCGGGCTGGAAG 23mer 76
human TRPV4 R315W_for GGCGGACATGTGGCGCCAGGACTC 24mer 78
human TRPV4 R315W_rev CGCGAGTCCTGGCGCCACATGTC 23mer 77
human TRPV4 R316C_for GACATGCGGTGCCAGGACTCGCG 23mer 77
human TRPV4 R316C_rev GCCTCGCGAGTCCTGGCACCGC 22mer 77
43
Chapter 2. Materials
Table 2.12: Oligonucleotides for Quickchange PCR
′ ′
Designation Sequence (5 –3 ) Length Tm [°C]
human TRPV4 I331F_for CTGGTGGCCTTTGCTGACAACACC 24mer 73
human TRPV4 I331F_rev GTTGTCAGCAAAGGCCACCAGCGCATG 27mer 77
human TRPV4 D333G_for CTGGTGGCCATTGCTGGCAACACCCGTG 28mer 80
human TRPV4 D333G_rev GTTCTCACGGGTGTTGCCAGCAATGGCCAC 30mer 81
human TRPV4 V342F_for CGTGAGAACACCAAGTTTTTTACCAAGATG 30mer 72
human TRPV4 V342F_rev GGTCGTACATCTTGGTAAAAAACTTGGTG 29mer 72
human TRPV4 M680K_for CTGACCATCGGCAAGGGCGAC 21mer 72
human TRPV4 M680K_rev CGCCCTTGCCGATGGTCAGC 20mer 72
Table 2.13: Oligonucleotides for Gibbson Assembly
′ ′
Designation Sequence (5 –3 ) Length Tm [°C]
pCAGIG_PACSIN1_V_for CTCAGATGGTTCCGCAGCACCAGTGGCCC 36mer 64
CGGCATG
pCAGIG_PACSIN1_V_rev CGTCCGTGGTACAGGTCGAGGATGCTACT 39mer 63
CCGGAGTGAC
pCAGIG_P1V_P3F_for CGATGAGGCCTCACTGATGGCTCCAGAA 38mer 62
GAGGACGCTG
pCAGIG_P1V_P3F_rev GCTGCGGAACCATCTGAGATCCTCT 42mer 63
TCGTCACTGGCTGCCTC
pCAGIG_P1V_P2F_for CGATGAGGCCTCACTGGTAGAAGTGTC 40mer 63
CAGCGACAGCTTCTG
pCAGIG_P1V_P2F_rev GCTGCGGAACCATCTGAGGTCCTCCAC 39mer 64
TGCATCAGCTGCTCTG
pCAGIG_PACSIN2_V_for CTGAGGTGGTTCCGAGCCAATCACGGG 36mer 65
CCGGGCATG
pCAGIG_PACSIN2_V_rev TCCAACGGAATCATCATATGTGACAGA 46mer 61
CATGGTGCCTGCTTTTTTG
44
Chapter 2. Materials
Table 2.13: Oligonucleotides for Gibbson Assembly
5′ 3′Designation Sequence ( – ) Length Tm [°C]
pCAGIG_P2V_P1F_for CATATGATGATTCCGTTGGAGCGCCAGA 41mer 65
GGAGACCACCGAC
pCAGIG_P2V_P1F_rev CTCGGAACCACCTCAGGTCTTCCTGGGC 37mer 64
pCAGIG_P2V_P3F_for CATATGATGATTCCGTTGGAATGGCTCC 43mer 64
AGAAGAGGACGCTGG
pCAGIG_P2V_P3F_rev CTCGGAACCACCTCAGATCCTCTTCGTC 40mer 63
ACTGGCTGCCTC
pCAGIG_PACSIN3_V_for CTGCGCTGGTGGCGCAGCACCCACGGGCC 32mer 64
AGG
pCAGIG_PACSIN3_V_rev GGTGGAGCCTGCTTTTTTGTACAAACTTG 48mer 62
TGATCAATTCGGTGCTGTC
pCAGIG_P3V_P1F_for AAAAAAGCAGGCTCCACCGCGCCAGAGGA 39mer 65
GACCACCGAC
pCAGIG_P3V_P1F_rev GCGCCACCAGCGCAGGTCTTCCTGGGCAT 37mer 64
CAGCCCCC
pCAGIG_P3V_P2F_for AAAAAGCAGGCTCCACCGTAGAAGTGTCC 39mer 64
AGCGACAGCTTCTGG
pCAGIG_P3V_P2F_rev GCGCCACCAGCGCAGGTCCTCCACTGCAT 40mer 63
CAGCTGCTCTG
Table 2.14: Sequencing oligonucleotides
′ ′
Designation Sequence (5 –3 ) Length
T7_promotor TAATACGACTCACTATAGGG 20mer
T7_terminator GCTAGTTATTGCTCAGCGG 19mer
SP6 ATTAGGTGACACTATAG 18mer
45
Chapter 2. Materials
2.6 Plasmids and expression constructs
T7_oligonucloetides (table 2.14 ) were used to determine the DNA sequence of the various
modified pET21, pET28a, pET11 and pcDNA3.1 plasmids (table 2.14). For reverse sequencing
of pcDNA3.1 the SP6 oligonucleotide was used. mm = Mus musculus (mouse), hs = Homo
sapiens (human), gg = Gallus gallus (chicken), rn = Rattus norvegicus (rat), d = Drosophila
melanogaster (fruit fly), dr = Danio rerio (zebra fish).
46
Chapter 2. Materials
47
Table 2.15: Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD, hsV4N∆122 as
hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the Hellmich workgroup.
Plasmid Construct Vector Resistance Origin
p17 eGFP pcDNA3.1 Ampicillin RG Mainz
p22 rnTRPV2-cGFP pcDNA3.1-cGFP Ampicillin Prof. , Cambridge
p24 ggTRPV4-cGFP pcDNA3.1-cGFP Ampicillin Prof. Cambridge
p25 dTRPML-cGFP pcDNA3.1-cGFP Ampicillin Prof. , Cambridge
p34 rnTRPV1-ARD pET21-cHis6 Ampicillin Prof. Cambridge
p35 drTRPV1-ARD pET21-cHis6 Ampicillin Prof. Cambridge
p36 ggTRPV1-ARD pET21-cHis6 Ampicillin Prof. Cambridge
p37 rnTRPV2-ARD pET21-cHis6 Ampicillin Prof. , Cambridge
p41 hsCalmodulin pET21-cHis6 Ampicillin Prof. Cambridge
p62 Ulp-1 pET28a Kanamycin Prof. , Frankfurt a.M.
p113 hsTRPML-1-nGFP pEGFP C3 Kanamycin Prof. , Cambridge
p114 mmTRPML-2b-c pcDNA3.1-cYFP Ampicillin Prof. , Santa Barbara
p129 ggV4N-cGFP pcDNA3.1-cGFP Ampicillin 21, Mainz
p130 ggV4N_aawaa-cGFP pcDNA3.1-cGFP Ampicillin i21, Mainz
p131 ggV4N∆53-cGFP pcDNA3.1-cGFP Ampicillin i21, Mainz
p132 ggV4N∆53_aawaa-cGFP pcDNA3.1-cGFP Ampicillin i21, Mainz
p134 hsV4N pET21-cHis6 Ampicillin Prof. , Cambridge (USA)
p138 BirA ligase pET21-cHis6 Ampicillin RG Mainz
p151 hsV4N Avi2 pET21-cHis6 Ampicillin B. Sc. E. Diehl, Mainz76
p154 hsV4N Avi3 pET21-cHis6 Ampicillin B. Sc. E. Diehl, Mainz76
p155 BirA ligase pET28a Kanamycin B. Sc. E. Diehl , Mainz76
Chapter 2. Materials
48
Table 2.15: Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD, hsV4N∆122 as
hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the Hellmich workgroup.
Plasmid Construct Vector Resistance Origin
p181 ggV4N∆122-cGFP pcDNA3.1-cGFP Ampicillin M. Sc. , Mainz
p182 ggV4N∆132-cGFP pcDNA3.1-cGFP Ampicillin M. Sc. , Mainz
p222 hsV4N∆132 pET21-cHis6 Ampicillin M. Sc. E. Diehl, Mainz76
p223 hsV4N∆122 pET21-cHis6 Ampicillin M. Sc. E. Diehl, Mainz76
p381 hsV4N∆132 E183K pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p382 hsV4N∆132 L199F pET21-cHis6 Ampicillin itt and M. Sc. E. Diehl, Mainz77
p383 hsV4N∆132 R232C pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p384 hsV4N∆132 R269C pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p385 hsV4N∆132 R271P pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p386 hsV4N∆132 K276E pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p387 hsV4N∆132 R315W pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p388 hsV4N∆132 R316C pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p389 hsV4N∆132 V342F pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p390 hsV4N∆132 Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl77, Mainz
p793 hsV4N∆132 E183K Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p794 hsV4N∆132 L199F Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p795 hsV4N∆132 R232C Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p796 hsV4N∆132 R269C Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p797 hsV4N∆132 R271P Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p798 hsV4N∆132 K276E Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p799 hsV4N∆132 R315W Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
Chapter 2. Materials
49
Table 2.15: Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD, hsV4N∆122 as
hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the Hellmich workgroup.
Plasmid Construct Vector Resistance Origin
p800 hsV4N∆132 R316C Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p801 hsV4N∆132 V342F Avi2 pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz77
p412 hsYAP1_WW1 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p413 hsYAP1_WW2 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p414 hsYAP1_WW1+WW2 domains pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p505 hsITCH pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p506 hsITCH_WW1 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p507 hsITCH_WW2 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p508 hsITCH_WW3 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p509 hsITCH_WW4 domain pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p510 hsITCH_WW1+WW2 domains pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p511 hsITCH_WW3+WW4 domains pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p512 hsRhoA pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p513 hsITCH_WW1−WW4 domains pET11a-nHis6 Ampicillin obtained from GeneScript, M. Sc. E. Diehl
p598 BirA R118A-nP1 pcDNA3.1-cGFP Ampicillin and M. Sc. E. Diehl, Mainz78
p802 hsTRPV4 pcDNA3.1-cGFP Ampicillin Prof. Baltimore
p803 hsTRPV4 S134E pcDNA3.1-cGFP Ampicillin and M. Sc. E. Diehl, Mainz78
p804 hsTRPV4 S134A pcDNA3.1-cGFP Ampicillin and M. Sc. E. Diehl, Mainz78
p805 hsTRPV4 R232C pcDNA3.1-cGFP Ampicillin this work
p806 hsTRPV4 R269C pcDNA3.1-cGFP Ampicillin this work
p807 hsTRPV4 R271P pcDNA3.1-cGFP Ampicillin this work
Chapter 2. Materials
50
Table 2.15: Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD, hsV4N∆122 as
hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the Hellmich workgroup.
Plasmid Construct Vector Resistance Origin
p808 hsTRPV4 K276E pcDNA3.1-cGFP Ampicillin this work
p809 hsTRPV4 R315W pcDNA3.1-cGFP Ampicillin this work
p810 hsTRPV4 V342F pcDNA3.1-cGFP Ampicillin this work
p811 hsTRPV4 M680K pcDNA3.1-cGFP Ampicillin and M. Sc. E. Diehl, Mainz78
p812 hsTRPV4 pcDNA3.1-cFLAG Ampicillin Prof. Baltimore
p813 hsTRPV4 R232C pcDNA3.1-cFLAG Ampicillin and M. Sc. E. Diehl,
Mainz77
p814 hsTRPV4 R269C pcDNA3.1-cFLAG Ampicillin Prof. , Baltimore
p815 hsV4N∆132 I331F pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz78
p816 hsV4N∆132 D333G pET21-cHis6 Ampicillin and M. Sc. E. Diehl, Mainz78
p817 hsDDX3X_aa122-582 pET21-nHis6 Kanamycin Prof. Regensburg
p818 hsDDX3X pcDNA3.1-nV5 Ampicillin obtained from GeneScript, M. Sc. E. Diehl,
Mainz
p819 mmPACSIN1 pCAGIG-nV5 Ampicillin Prof. Baltimore
p820 mmPACSIN2 pCAGIG-nV5 Ampicillin Prof. Baltimore
p821 mmPACSIN3 pCAGIG-nV5 Ampicillin Prof. Baltimore
p822 hsPACSIN1 pCAGIG-nV5 Ampicillin Prof. , Baltimore
p823 hsPACSIN2 pCAGIG-nV5 Ampicillin Prof. , Baltimore
p824 hsPACSIN3 pCAGIG-nV5 Ampicillin Prof. Baltimore
p825 hsPACSIN_F1_S2 pCAGIG-nV5 Ampicillin this work
(hsPACSIN2+1)
Chapter 2. Materials
51
Table 2.15: Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD, hsV4N∆122 as
hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the Hellmich workgroup.
Plasmid Construct Vector Resistance Origin
p826 hsPACSIN_F1_S3 pCAGIG-nV5 Ampicillin this work
(hsPACSIN3+1)
p827 hsPACSIN_F2_S1 pCAGIG-nV5 Ampicillin this work
(hsPACSIN1+2)
p828 hsPACSIN_F2_S3 pCAGIG-nV5 Ampicillin this work
(hsPACSIN3+2)
p829 hsPACSIN_F3_S1 pCAGIG-nV5 Ampicillin this work
(hsPACSIN1+3)
p830 hsPACSIN_F3_S2 pCAGIG-nV5 Ampicillin this work
(hsPACSIN2+3)
p831 mCherry pcDNA3.1 Ampicillin Prof. Baltimore
p832 hsYAP1 pcDNA3.1-nV5 Ampicillin Prof. , Baltimore
p833 hsRUVBL1 pcDNA3.1-nmyc Ampicillin Prof. Baltimore
p834 Empty Vector (EV) pcDNA3.1 Ampicillin Prof. Baltimore
p835 hsTRPC3 ARD_aa16-181 pET11a-nHis6 Ampicillin Prof. , Cambridge
p836 hsTRPC4 ARD_aa25-185 pET11a-nHis6 Ampicillin Prof. Cambridge
p837 hsTRPC6 ARD_aa87-252 pET11a-nHis6 Ampicillin Prof. , Cambridge
Chapter 2. Materials
2.7 Peptide and protein characteristics
In the following, protein and peptide characteristics of the constructs used in this work are listed.
Table 2.16 lists all constructs which were recombinantly expressed in E. coli BL21 Gold (DE3)
cells and subsequently purified according section 3.7. Characteristics of protein and peptides
with no TEV-cleavage site are listed with the His6-tag, whereas TEV cleavage site containing
proteins are listed without His6-tag.
Table 2.17 lists all protein construct which were recombinantly expressed in HEK293 and/or
HEK293T cells (see also sections3.2.1, 3.3, 3.4 and 3.5).
Table 2.16: Physicochemical properties of proteins and peptides recombinantly expressed in E.
coli BL21 Gold (DE3), purified and used in this thesis. Characteristics were calculated using
the Protparam tool (http://web.expasy.org/protparam/). hsV4N∆132 is also referred to as hsV4
ARD, hsV4N∆122 as hsV4 ARD-PRR. MW: molecular weight [Da]; AA: number of amino acids;
pI: isoelectric point;  280: extinction coefficient at 280 nm in [L mol−1 cm−1]
Peptide/ protein MW [Da] AA pI  280
hsDDX3X_aa 122-582 52 057 461 5.61 44 725
BirA ligase 35 312 321 7.76 47 440
hsV4N-cHis6 44 977 406 8.24 20 650
hsV4N Avi2-cHis6 46 788 421 7.00 25 900
hsV4N∆122-cHis6 30 616 271 9.00 10 680
hsV4N∆132-cHis6 29 221 257 8.65 10 680
hsV4N∆132-cHis6 R232C 29 221 257 8.65 10 680
hsV4N∆132-cHis6 R269C 29 221 257 8.65 10 680
hsV4N∆132-cHis6 D333G 29 221 257 8.65 10 680
hsV4N∆132-Avi2 31 089 273 7.39 16 180
hsV4N∆132-Avi2 R232C 31 089 273 7.39 16 180
hsV4N∆132-Avi2 K276E 31 089 273 7.39 16 180
hsV4N∆132-Avi2 V342F 31 089 273 7.39 16 180
hsTRPC3 ARD_aa16-181-nHis6 19 887 175 5.49 10 555
hsTRPC4 ARD_aa25-185-nHis6 18 055 161 5.91 7450
hsTRPC6 ARD_aa87-252-nHis6 19 923 175 5.55 10 555
hsYAP1_WW1 domain 5184 45 4.89 12 490
52
Chapter 2. Materials
Peptide/ protein MW [Da] AA pI  280
hsYAP1_WW2 domain 4589 40 4.89 13 980
hsYAP1_WW1+WW2 domains 11 926 105 4.88 26 470
hsITCH 102 859 904 5.94 154 085
hsITCH_WW1 domain 14 112 135 10.34 13 980
hsITCH_WW2 domain 4246 35 8.59 13 980
hsITCH_WW3 domain 3981 34 8.59 12 490
hsITCH_WW4 domain 4923 42 5.58 8480
hsITCH_WW1+WW2 domains 17 926 166 10.48 27 960
hsITCH_WW3+WW4 domains 8772 74 6.94 20 970
hsITCH_WW1-WW4 domains 32 219 286 9.59 57 410
hsRhoA 21 825 194 5.84 18 825
Table 2.17: Physicochemical properties of proteins and peptides recombinantly expressed in
HEK293 and/or HEK293T cells in this thesis. Characteristics were calculated using the Prot-
param tool (http://web.expasy.org/protparam/). MW: molecular weight [Da]; AA: number of
amino acids
Protein MW [Da] AA
hsTRPV4-cGFP 125 204 1110
hsTRPV4 R232C-cGFP 125 204 1110
hsTRPV4-nFLAG 99 276 879
hsTRPV4 R269C-nFLAG 99 276 879
hsPACSIN-1-nV5 53 776 471
hsPACSIN-2-nV5 58 549 513
hsPACSIN-3-nV5 51 297 451
hsPACSIN-F1-S2-nV5 34 879 305
(hsPACSIN2+1-nV5)
hsPACSIN-F1-S3-nV5 40 859 355
(hsPACSIN3+1-nV5)
hsPACSIN-F2-S1-nV5 53 423 471
53
Chapter 2. Materials
Protein MW [Da] AA
(hsPACSIN1+2-nV5)
hsPACSIN-F2-S3-nV5 50 823 444
(hsPACSIN3+2-nV5)
hsPACSIN-F3-S1-nV5 54 155 479
(hsPACSIN1+3-nV5)
hsPACSIN-F3-S2-nV5 36 478 318
(hsPACSIN3+2-nV5)
hsDDX3X-nV5 76 154 690
hsRUVBL1-myc 51 757 470
mCherry 26 722 236
2.8 Kits
Table 2.18: Kits
Designation Usage Supplier
E.Z.N.A Plasmid Mini Kit I Plasmid DNA isolation Omega Bio-Tek, Norcross (USA)
GenepHlow Gel/PCR Kit PCR clean up Geneaid, New Tapei City (TW)
GTPase Glo Assay Instrinsic GTPase
acivity assay Promega, Walldorf
KAPA HiFi HotStart PCR Kit PCR ingredients Kapa Biosystems, Wilmington (USA)
Fluo-4 Direct Calcium Assay Ca2+-influx assay Invitrogen, Darmstadt
4 - 15 % Mini-Protean® TGX™ Blue native (BN)
Precast Protein Gels PAGE gels BioRad, Hercules (USA)
54
Chapter 2. Materials
2.9 Media and supplements for cell culture
Table 2.19: Media and supplements for cell culture
Name Supplier
LB-Medium (Luria Broth) Roth, Karlsruhe
TB-Medium (Terrific Broth) Roth, Karlsruhe
Dulbecco’s Modified Eagle Medium (DMEM) Invitrogen, Darmstadt
Fetal calf serum (FCS) Invitrogen, Darmstadt
10x Trypsin Invitrogen, Darmstadt
G-418 Sigma-Aldrich, Munich
L-Glutamine Invitrogen, Darmstadt
Penicillin / Streptomycin (P/S) Sigma-Aldrich, Munich
Poly-L-lysine Invitrogen, Darmstadt
2.10 Cells
Cells were obtained from various sources indicated in table 2.20. HEK293-hsV4-cGFP and
HEK293-hsTRPML1-nGFP are stably transfected HEK293 cell strains which were obtained in
the course of this work (see 3.2.3 for procedure).
Table 2.20: Cells
Name Source
E. coli strain BL21 (DE3) Gold Agilent Technologies, Santa Clara (USA)
E. coli strain DH5α Agilent Technologies, Santa Clara (USA)
HEK293 cells PD , Mainz
HEK293T cells Prof. , Mainz
Prof. Baltimore (USA)
HEK293-hsV4-cGFP this work
55
Chapter 2. Materials
Table 2.20: Cells
Name Source
HEK293-hsTRPML1-nGFP this work
2.11 Laboratory equipment
Table 2.21: Laboratory equipment
Name Supplier
Acquity UPLC HSS-T3 reverse phase column Waters Corporation, Milford (USA)
Avanti J-26XP Centrifuge Beckmann Coulter, Brea (USA)
Bruker AVANCE 600, 700 and 800 MHz spectrometers Bruker, Karlsruhe
Branson Sonifier 250 Branson, Danbury (USA)
Centrifuge 5810 R Eppendorf, Hamburg
Centrifuge 5415 R Eppendorf, Hamburg
Centrifuge rotor JA 25.50 Eppendorf, Hamburg
Centrifuge rotor JLA 8.1000 Eppendorf, Hamburg
Optima MAX XP Ultra-centrifuge Beckmann Coulter, Brea (USA)
NGC-Quest ÄKTA purifier BioRad, Hercules (USA)
FlexStation 3 MultiMode Plate Reader MolecularDevices, San Jose (USA)
FLUOStar Omega Microplate Reader BMG LabTech, Ortenberg
Frac-920 Fraction collector BioRad, Hercules (USA)
INCU-line IL 10 Incubator VWR, Radnor (USA)
J-815 CD Spectrometer JASCO, Pfungstadt
Lamda DG-4 wavelength switcher Sutter Instruments, San Francisco (USA)
pH electrode LE409 Mettler-Toledo, Columbus (USA)
Photometer Lambda 25 Perkin Elmer , Waltham (USA)
PowerPac Basic power supply BioRad, Hercules (USA)
ViewPix700 Scanner GE Healthcare, Fairfield (USA)
56
Chapter 2. Materials
Name Supplier
Mini-PROTEAN Tetra Cell BioRad, Hercules (USA)
HiLoad 16/600 Superdex 75 SEC column GE Healthcare, Fairfield (USA)
HiLoad 16/600 Superdex 200 SEC column GE Healthcare, Fairfield (USA)
Spectrofluorometric detector RF-20A Shimadzu, Kyoto (Japan)
STELLA 3200 CCD camera Raytest GmbH, Straubenhardt
Synapt G2-S HDMS Waters Corporation, Milford (USA)
Thermomixer comfort Eppendorf, Hamburg
nanoAcquity UPLC Waters Corporation, Milford (USA)
NanoDrop 2000c UV-Vis spectrophotometer Thermo Scientific, Waltham (USA)
Membrane pump MD 4C Vacuubrand, Wertheim
Thermal cycler Primus 25 MWG Biotech, Ebersberg
Quantum gel documentation Peqlab, Erlangen
ViewPix 700 Scanner Biostep, Burkhardtsdorf
Zeiss Axio Observer Z1 Inverted Microscope Zeiss, Jena
Zeiss LSM800 Confocal Microscope Zeiss, Jena
2.12 Software
Table 2.22: Software
Name Source / distributor
Adobe Photoshop CS 5 Adobe Systems GmbH, München
CARA AG Wüthrich, ETH Zürich (CH)
http://cara.nmr.ch/
CorelDraw 2019 Corel Corporation, Ottawa (CAN)
CytoScape Cytoscape Consortium
TEXStudio Free Software Foundation
DrawTree 3.66 LIRMM, Montpellier (FRA)
David Go Laboratory of Human Retrovirology
57
Chapter 2. Materials
Name
and Immunoinformatics, Frederick (USA)
BibLATEX Free Software Foundation
TEXlive Free Software Foundation
ProtParam ExPasy, SIB Swiss Institute of Bioinformatics
TranslateTool ExPasy, SIB Swiss Institute of Bioinformatics
TOPSPIN 3.5 pl 5 Bruker, Karlsruhe
Clustal Omega EMBL-EBI, Hinxton (GB)
CAPITO Fritz Lipmann Institute, Leipzig
OriginLab 7 OriginLab, Northampton (USA)
58
Chapter 3. Methods
3 Methods
3.1 General methods
3.1.1 Heat-shock transformation of E. coli bacteria
0.5 µL of plasmid DNA were added to 25 µL of competent E. coli BL21 (DE33) Gold or DH5α
cells and incubated on ice for 30 min. Cells were heat shocked at 42 °C for 45 s and again
incubated on ice for 2 min. Afterwards, 300 µL of LB-Medium were added and the cells were
grown for 1 h at 37 °C and 225 rpm. Subsequently, the cells were plated onto an antibiotic
containing agar plate and incubated over night at 37 °C.
3.1.2 Plasmid DNA preparation from E. coli
An over night culture of E. coli DH5α cells was cultivated in 5 mL LB medium at 37 °C which
contained the suitable amount of antibiotics. After centrifugation at 3900 rpm for 10 min at
4 °C the plasmid DNA was isolated with the E.Z.N.A Plasmid Mini Kit I (protocol I of instruction
manual). DNA concentration was determined by absorption measurements at 260 nm with a
ThermoScientific NanoDrop 2000c UV-Vis spectrophotometer.
3.1.3 Polymerase chain reaction (PCR)
Modifications and amplifications of DNA fragments were performed via PCR. For this the Kapa
HiFi HotStart PCR Kit and a Primus 25 thermo cycler were used. For more details on used
oligonucleotides and methods see subsection 3.1.4 and tables 2.13 and 2.12. Prior to further
usage, the template DNA was digested with DpnI, which specifically recognizes methylated
59
Chapter 3. Methods
DNA. Furthermore the DNA was cleaned up with the GenepHlow Gel/PCR Kit and eluted in
30 µL ddH2O.
Table 3.1: DpnI digestion reaction mixture
Component Volume [µL]
10x Cutsmart buffer 5
ddH2O 22.5
DpnI 1
PCR reaction mixture 22.5
3.1.4 Gibson Assembly
Gibson Assembly is a suitable method to merge linear DNA fragments to obtain, for example,
new combinations of insert and vector DNA.79,80 After generation of linear DNA segments via
PCR, annealing of the fragments is performed in a buffer which contains three different en-
zymes. First, an exonuclease creates single-stranded 3’ overhangs for annealing purposes.
The occurring gaps in the annealed DNA fragments are filled by a DNA polymerase and a DNA
ligase covalently links the fragments. After incubation of the DNA fragments in the isothermal
ligation reaction mix (see table 3.2) for 1 h at 50 °C, the DNA was transformed into E. coli DH5α
cells and isolated as described in sections 3.1.1 and 3.1.2.
Table 3.2: Reaction mixtures for Gibson Assembly
Buffer/solution Ingredients
5x Isothermal Reaction Mix 0.5 M Tris-HCl pH = 7.5
0.5 M MgCl2
1 mM dNTP Mix
0.25 g/mL PEG-8000
0.5 M NAD+
Assembly Master Mix 1x Isothermal Master Mix
60
Chapter 3. Methods
Buffer/solution Ingredients
0.5 U/µL T5 Exonuclease
0.3 U/µL Phusion DNA Polymerase
0.5 U/µL Taq DNA Ligase
3.1.5 Agarose gel electrophoresis
To separate DNA with various sizes agarose gel electrophoresis was performed. 1 or 2 %
(w/v) agarose gels were used. Visualization of the different DNA plasmids and fragments was
achieved by ethidium bromide staining for at least 20 min and detection with UV-light.
3.1.6 DNA sequencing
To confirm successful cloning of genes, DNA sequencing was performed by GENterprise GE-
NOMICS (StarSEQ GmbH, Mainz). The primers from table 2.14 were used. The final sequenc-
ing mixture contained 300 - 700 ng plasmid DNA and 1 µL sequencing primer (10 µM stock solu-
tion, see also table 2.14) topped up to a total volume of 7 µL with ddH2O.
3.1.7 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
Proteins were separated according to their molecular weight with SDS-PAGE. For buffer compo-
sitions see table 2.2. Amongst other methods (see also section 3.1.8), visualization of the pro-
tein bands were accomplished by Coomassie staining. For the compositions of the Coomassie
staining and destaining solution also see table 2.2.
Table 3.3: Composition of the stacking and separation gel for SDS-PAGE
Ingredients separation gel 15 % separation gel 12 % stacking gel
ddH2O 4.6 mL 4.6 mL 4.6 mL
1.5 mM Tris-HCl pH 8.8 5 mL 5 mL /
61
Chapter 3. Methods
Table 3.3: Composition of the stacking and separation gel for SDS-PAGE
Ingredients separation gel 15 % separation gel 12 % stacking gel
1.0 mM Tris-HCl pH 6.8 / / 0.63 mL
Acrylamid/Bisacrylamid 30 % 10 mL 8 mL 0.83 mL
SDS 10 % (w/v) 0.2 mL 0.2 mL 0.5 mL
APS 10 % (w/v) 0.2 mL 0.2 mL 0.5 mL
TEMED 0.2 mL 0.2 mL 0.5 mL
3.1.8 Western blot and chemiluminescence detection
After SDS-PAGE (see section 3.1.7) separated proteins were transferred onto a PVDF mem-
brane with another electrophoresis step to detect specific proteins and/or their modifications,
respectively, with an antibody mediated chemiluminescence reaction. Depending on the used
antibodies, the membrane was incubated in different blocking solutions for 1 h at room temper-
ature (see also 3.4). After three wash steps, each for 5 min at room temperature with PBS/T,
the membrane was incubated in the particular peroxidase-coupled antibody solution (see also
3.5). Again, three wash steps were performed and chemiluminescence was induced with a
1:1-mixture of Roti®-Lumin reagents 1 and 2 detected either with a STELLA 3200 or GE Image-
Quant CCD camera.
Table 3.4: Used primary antibody solutions and blocking solutions
Antibody solution Blocking solution (% in w/v)
Anti-His6-HRP 1:5000 in 2 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Strep-POD 1:20 000 in 1 % (w/v) BSA in PBS/T 2 % (w/v) BSA in PBS/T
Anti-ANXA2 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Anti-DDX3X 1:1000 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Anti-V5 mouse 1:5000 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Anti-myc mouse 1:1000 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Anti-FLAG rabbit 1:1000 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
Anti-FLAG mouse 1:1000 in 1 % (w/v) milk powder in PBS/T 5 % (w/v) milk powder in PBS/T
62
Chapter 3. Methods
Table 3.5: Used secondary antibody solutions
Antibody solution
Horse anti-mouse HRP-linked IgG 1:1000 in PBS/T
Goat anti-rabbit HRP-linked IgG 1:1000 in PBS/T
3.1.9 Protein quantification
To determine the protein concentrations various methods were used.
Bradford assay
With the Bradford Assay it is possible to obtain the concentration of protein in a sample via
the quantitative assessment of the absorbance shift of Coomassie G-250 based on interactions
between dye and proteins. As standards, increasing concentrations of BSA solutions were used
(0 to 200 µg/µL).
Absorption measurements
Another method to assess the protein concentration of a sample is to measure the absorption
at 280 nm with a ThermoScientific NanoDrop 2000c UV-Vis spectrophotometer due to the ab-
sorption of aromatic amino acids (tyrosine, tryptophan, phenylalanine). Because the number
of these aromatic amino acids are varying in each protein, the specific molar extinction coeffi-
cients at 280 nm were computed using the ExPasy-ProtParam tool (http://web.expasy.org/
protparam/).
63
Chapter 3. Methods
3.1.10 Size exclusion chromatography
Proteins were purified according to their size via size exclusion chromatography (SEC). Depend-
ing on the sample volume and protein size, various columns were used (see table 3.6)
Table 3.6: Used SEC columns, manufactured by GE Healthcare
Column name column volume sample volume separation range
HiLoad 16/600 Superdex 200 120 mL ≤ 5 mL 70 000 - 3000 Da
HiLoad 16/600 Superdex 75 120 mL ≤ 5 mL 600 000 - 10 000 Da
Superose™ 6 10/300 GL 24 mL ≤ 500 µL 5 000 000 - 5000 Da
Columns were stored at 4 °C in 20 % (v/v) ethanol. SEC runs for each protein were also car-
ried out at 4 °C and the columns were equilibrated in the appropriate SEC buffer before every
protein purification. Chosen protein samples were adjusted to the desired sample volume with
a centrifugal concentrator. Potential aggregates were removed by centrifugation at 3900 rpm
and 4 °C for 10 min before loading it onto the SEC column. The sample was loaded onto a
suitable loop and injected onto the column with a flow rate of 0.5 mL/min. Elution was per-
formed isocratically for 1.1 CV. The flow rates during elution as well as the collected fraction
size were adjusted to the individual proteins (see 3.7). The absorbence at 280 nm was de-
tected and the purity of collected fractions verified via SDS-PAGE with subsequent Coomassie
staining.
3.2 Cell culture of eukaryotic cells
3.2.1 Cultivation
HEK293 cell strains (see table 2.20) were cultivated in 10 cm dishes at 37 °C, 5 % CO2 atmo-
sphere and 95 % humidity with DMEM containing 10 % (v/v) FCS, 1 % (v/v) P/S and 1 % (v/v) L-
Glutamine. HEK293-hsV4-cGFP were additionally treated with 0.500 µg/mL G-418 and 200 nM
64
Chapter 3. Methods
TRPV4 inhibitor HC-067, HEK293-hsTRPML1-nGFP cells with 0.375 µg/mL G-418. Twice a
week the cells were splitted in a ratio of 1:8. For splitting, the expended medium was aspi-
rated and the cells were washed with 5 mL PBS. Afterwards, the cells were trypsinized with
1.5 mL trypsin/EDTA in 1.5 mL PBS and diluted to the desired ratio with fresh medium. The cells
were then plated on fresh dishes. All media were warmed up to 37 °C in a water bath before
use.
3.2.2 Transient transfection of HEK293T cells
For transient transfection of HEK293T cells, 575 000 cells were seeded into 6-well plates per
well 24 h before transfection. Cells were transfected according the Lipofectamin® LTX reagent
protocol. For fluorescence microscopy (see section 3.3) and live calcium imaging (see 3.5) poly-
L-lysinated cover slips were provided in the 6-well dish. Used plasmid DNAs are listed in table
2.15 and used plasmid DNA amounts for transfections in table 3.7. If not otherwise indicated,
cells were incubated with the DNA/lipid complex for 24 h.
Table 3.7: Used plasmid DNA amounts for transfections of HEK293T cells with Lipofectamine
® LTX
Expressed construct Vector Plasmid DNA amount [ng/well]
ggV4N-cGFP pcDNA3.1-cGFP 2000
ggV4N_aawaa-cGFP pcDNA3.1-cGFP 2000
ggV4N∆53-cGFP pcDNA3.1-cGFP 2000
ggV4N∆53_aawaa-CGFP pcDNA3.1-cGFP 2000
hsTRPV4-cGFP pcDNA3.1-cGFP 500
hsTRPV4 S134E-cGFP pcDNA3.1-cGFP 500
hsTRPV4 S134A-cGFP pcDNA3.1-cGFP 500
hsTRPV4 R232C-cGFP pcDNA3.1-cGFP 500
hsTRPV4 R269C-cGFP pcDNA3.1-cGFP 500
hsTRPV4 K276E-cGFP pcDNA3.1-cGFP 500
hsTRPV4 R315W-cGFP pcDNA3.1-cGFP 500
hsTRPV4 V342F-cGFP pcDNA3.1-cGFP 500
hsTRPV4 M680K-cGFP pcDNA3.1-cGFP 500
hsTRPV4-nFLAG pcDNA3.1-nFLAG 500
65
Chapter 3. Methods
Expresssed construct Vector Plasmid DNA amount [ng/well]
hsTRPV4-R269C-nFLAG pcDNA3.1-nFLAG 500
hsDDX3X-nV5 pcDNA3.1-nV5 1000
mmPACSIN1-nV5 pCAGIG-nV5 1000
mmPACSIN2-nV5 pCAGIG-nV5 1000
mmPACSIN3-nV5 pCAGIG-nV5 1000
hsPACSIN1-nV5 pCAGIG-nV5 500
hsPACSIN2-nV5 pCAGIG-nV5 500
hsPACSIN3-nV5 pCAGIG-nV5 500
hsPACSIN_F1_S2-nV5 pCAGIG-nV5 1000, 48 h
hsPACSIN_F1_S3-nV5 pCAGIG-nV5 1000, 48 h
hsPACSIN_F2_S1-nV5 pCAGIG-nV5 1000, 48 h
hsPACSIN_F2_S3-nV5 pCAGIG-nV5 1000, 48 h
hsPACSIN_F3_S1-nV5 pCAGIG-nV5 1000, 48 h
hsPACSIN_F3_S2-nV5 pCAGIG-nV5 1000, 48 h
mCherry pcDNA3.1 500
hsYAP1-nV5 pcDNA3.1-nV5 500
hsRUVBL1-myc pcDNA3.1-nmyc 500
3.2.3 Stable transfection of HEK293 cells
To generate stable HEK293 cell lines stably expressing either hsTRPV4-cGFP or hsTRPML1-
nGFP, HEK293 cells were transfected as previoulsy described (see 3.2.2). For used plas-
mid DNA amounts, see table 3.8. Furthermore, after transfection, HEK293 cells expressing
hsTRPV4-cGFP (HEK293-hsV4-cGFP), if not otherwise indicated, were always treated with
200 nM of the TRPV4 antagonist HC-067.
66
Chapter 3. Methods
Table 3.8: Used plasmid amounts for stable transfections of HEK293 cells with Lipofectamine
® LTX
Expressed construct Vector Plasmid DNA amount [ng/well] G-418 amount [µg/µL]
hsTRPV4-cGFP pcDNA3.1-cGFP 500 0.500
hsTRPML1-nGFP pEGFP C3 2000 0.375
After 24 h incubation with the DNA/lipid complex, cells were examined for GFP-signals on a
widefield Zeiss Axio Observer Z1 Inverted Microscope. If GFP-positive, cells were passaged
for 2 weeks and selected with G-418 (see table 3.8), with G-418-containing media changes
after every 48 h. Large, healthy and GFP-positive colonies were isolated with sterile tooth-
picks and cultured for another 3 weeks in G-418 medium with media changes after every 48 h.
With this, stably expressing, polyclonal cell lines were obtained. Proper protein expression,
function and localization were confirmed with Western Blots with subsequent chemilumines-
cence detection, fluorescence microscopy and Ca2+-influx assays. Generation of monoclonal
cell lines failed, as the single cell transfer into 96-well plates resulted multiple times in cell
death.
3.3 Immunostaining and fluorescence microscopy
HEK293T or HEK293 cells were stably or transiently transfected with plasmid DNA encoding for
the desired protein (see sections 3.2.2 and 3.2.3). To obtain insights in protein localization and
protein-protein co-localization in cells, immunostaining was performed on the cells. Cells were
washed three times with PBS containing 0.2 % (v/v) Tween®-20 (PBS/T), followed by an incuba-
tion step with 50 mM NH4Cl in PBS for 10 min due to formaldehyde quenching. To permeabilize
the cell membranes towards the staining antibodies or reagents, the cells were incubated in
PBS containing 0.3 % (v/v) Triton-X for 15 min at RT and washed with PBS for 3 times. After
discarding, the cells were incubated in 1 % (w/v) BSA in PBS/T for 30 min and washed with
PBS/T for three times. If needed, primary antibodies diluted in 1 % (w/v) BSA in PBS/T (see
table 3.9) were applied to the cells and incubated at 4 °C over night. After washing with PBS/T
for three times, respective secondary antibodies in PBS/T were applied and incubated for 1 h
67
Chapter 3. Methods
at RT. Again three washing steps with PBS/T were performed. For F-actin staining, cells were
incubated for 1 h with Phalloidin-TRITC at RT and after 3 PBS/T washing steps, nuclei were
stained with DAPI for 30 min at RT. The cells were washed again for three times with PBS/T
and finally with pure PBS. The cell bearing cover slips were mounted on microscopic slides with
Mowiol® 4-88 and kept in the dark until further usage.
Table 3.9: Antibodies used for cell immunostainings
Antibody Amount [µg/mL]
Anti-ANXA2 mouse 2
Anti-DDX3X rabbit 2
Anti-FLAG rabbit 2
Anti-V5 mouse 2.5
Anti-myc mouse 2
Alexa-Fluor-488 goat anti-mouse 4
Alexa-Fluor-555 goat anti-rabbit 4
Alexa Fluor-647 goat anti-mouse 5
Alexa Fluor-647 goat anti-rabbit 5
Phalloidin-TRITC 50
Fluorescence microscopic images were obtained with a wide field Zeiss Axio Observer Z1 In-
verted Microscope, kindly provided by Prof. Gerald Gimpl (Department of Chemistry, Biochem-
istry Section, Johannes Gutenberg University, Mainz), or a Zeiss LSM800 Confocal Microscope
(Microscopy Core Facility, JHU School of Medicine). The obtained pictures were extracted and
processed with the biological-image analysis program Fiji.81
3.4 Co-Immunoprecipitation in eukaryotic cells
Co-Immunoprecipitation (co-IP) is a straight-forward technique to evaluate protein complexes
under physiological conditions. In this work, an antibody-based co-IP protocol with protein G-
coated magnetic beads was performed. Transfected cells (see sections 3.2.2 and 3.2.3) were
68
Chapter 3. Methods
washed three times with ice-cold PBS and then harvested with 1 mL/well of a 6-well plate. Cells
were lysed with Pierce™Lysis Buffer supplemented with CST Protease/Phosphatase-Inhibitor.
After two centrifugation steps at 4600 rpm and 13 900 rpm, respectively, for 15 min at 4 °C, the
supernatant was transferred in a fresh, pre-chilled tube. The Protein G-coupled beads were
coated with respective antibody for 15 min at RT and then were washed one time with PBS/T.
After this, the antibody-coated beads were incubated with the supernatant for 2 h at 4 °C under
mild rotation. Beads were then washed three times with PBS/T and finally, proteins were eluted
from the beads with 1x SDS-PAGE sample buffer (see table 2.2) at 70 °C for 10 min. Samples
then were subjected to SDS-PAGE and western blotting with subsequent chemiluminescence
detection (see sections 3.1.7 and 3.1.8).
3.5 Live Ca2+ imaging
Live calcium imaging (Ca2+ imaging) was performed on a Zeiss Axio Observer Z1 inverted
microscope equipped with a Lambda DG-4 wavelength switcher. 24 h prior treatment, 6.8x105
cells/well were seeded int a 6-well plate, provided with Poly-L-coated cover slips in each well.
1 h prior treatment and Ca2+ imaging, cells were loaded with 2.5 µM Fura2 AM for 60 min at
37 °C. For hypotonic treatment, three volumes of hypotonic aCSF (see 2.2) was added to one
volume of aCSF (see table 2.2 for buffer composition). For GSK-101 treatment, GSK-101 was
added directly to the aCSF buffer. Cells were imaged every 10 seconds for 1 min prior to
stimulation with hypotonic saline or GSK-101, then imaged every 10 seconds for an additional
8 min. Calcium levels at each time point were computed by determining the ratio of Fura-2 AM
emission at 380 nm divided by the emission at 340 nm. Data are respresenting raw the Fura
ratio.
3.6 Ca2+ influx assay
To assure the functionality of stably transfected human TRPV4-cGFP (see also section 3.2.3),
fluorescence-based Ca2+ influx assays were performed with the Fluo-4 Direct Calcium Assay
on a FlexStation 3 MultiMode Plate Reader. Poly-L-lysinated 96-well plates were seeded with
40 000 cells/well and after 24 h, cells were loaded with Fluo-4 Direct™calcium reagent loading
69
Chapter 3. Methods
solution according to the manufacturers protocol. After incubation for 1 h at RT, 5 % CO2 atmo-
sphere and 95 % humidity, cells were treated with 30 nM GSK-101 end concentration. Resulting
fluorescence signals were measured every 3 s for 3 min at RT with an excitation wavelength at
494 nm, an emission wavelength 516 nm and a bandwidth of 1 nm.
3.7 Recombinant expression and purification of constructs
3.7.1 Recombinant expression of human N-terminal TRPV4 (hsV4N) constructs
E. coli BL21 (DE3) Gold cells were heat-shock transformed (see section 3.1.1) with the respec-
tive plasmid (see table 2.15). In an over night incubation at 37 °C and 225 rpm a preculture in
50 mL LB medium containing 100 µg/mL ampicillin was set up. Subsequently, 1 L TB medium
with 0.2 % (v/v) glucose or LB medium were supplemented with 100 µg/mL ampicillin and inoc-
ulated with 2 % (v/v) of preculture. The cells were then grown at 37 °C and 225 rpm to an OD600
of 0.6. Used IPTG concentrations and conditions for protein expression are listed in table 3.10.
The cells were harvested by centrifugation at 5000 rpm and 4 °C for 10 min. After disposing the
supernatant, the cell pellet was frozen with liquid nitrogen and stored at −20 °C until further
use.
Table 3.10: Expression conditions of human N-terminal TRPV4 (hsV4N) constructs.
hsV4N∆132 is also referred to as hsV4 ARD, hsV4N∆122 as hsV4 ARD-PRR. Avi2 = c-terminal
avidin-tag followed by a His6-tag. IPTG conc. = IPTG concentration, Temp. = Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
hsV4N-His6 TB 150 16 20
hsV4N R232C-His6 LB 150 16 20
hsV4N-Avi2 TB 150 16 20
hsV4N∆122-His6 LB 75 16 20
hsV4N∆132-His6 LB 75 18 20
hsV4N∆132 R232C-His6 LB 75 18 20
hsV4N∆132 R269C-His6 LB 75 18 20
hsV4N∆132 D333G-His6 LB 75 18 20
hsV4N∆132-Avi2 LB 75 16 20
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Chapter 3. Methods
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
hsV4N∆132 R232C-Avi2 LB 75 18 20
hsV4N∆132 K276E-Avi2 LB 75 18 20
hsV4N∆132 V342F-Avi2 LB 75 18 20
The below mentioned purifications were all carried out at 4 °C.
3.7.2 Purification of hsV4N constructs
The cell pellet of a 1 L E. coli cell culture was suspendend in 100 mL E. coli lysis buffer (for all
buffer compositions see 2.2). Sonification was performed on ice with a Branson Sonifier 250
using a duty cycle of 5 for 50 s. The cell lysate was cleared via centrifugation at 20 000 rpm and
4 °C for 40 min. The obtained supernatant was loaded onto 1 mL Ni2+-NTA agarose provided in
a column which was equilibrated with 2 CV Ni2+-NTA buffer 20 beforehand. The flow through
was discarded and the Ni2+-NTA agarose then washed with 20 CV Ni2+-NTA buffer 20, followed
by a second and a third wash step with 1 CV Ni2+-NTA buffer 50 and 1 CV Ni2+-NTA buffer 75,
respectively. The His6-tagged protein was eluted with 9 CV Ni2+-NTA buffer 500 and collected
in 1 mL fractions, to which 1 mM DTT and 1 mM EDTA were added. Protein purity was analyzed
via SDS-PAGE using a 12 % gel. The protein was further subjected to size exclusion chromatog-
raphy with a HiLoad 16/600 Superdex 200 SEC column, whereas elution was performed over
1.1 CV with SEC buffer hsV4N and collected in 0.5 mL fractions. If necessary, a second SEC
with the before purified fractions was performed with SEC buffer hsV4N containing 750 mM
NaCl. Purity was analyzed via SDS-PAGE and Coomassie staining.
3.7.3 Purification of hsV4∆N122 and hsV4∆N132 constructs
hsV4N∆132 is also referred to as hsV4 ARD, hsV4N∆122 as hsV4 ARD-PRR. The same
purification protocol as described in subsection 3.7.2 above was used. The last wash step
(W3) was left out, also 10 elution steps with Ni2+-NTA buffer 500 were performed. For human
RhoA interaction studies via NMR with 15N-labeled human RhoA (see section 4.4), the SEC of
hsV4N∆132 R232C-His6 was performed with the SEC buffer of RhoA (see table 2.4).
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Chapter 3. Methods
3.7.4 Biotinylation and purification of hsV4N constructs
With the insertion of the 15 amino acids containing (GLNDIFEAQKIEWHE) avidin tag (Avi) in
the protein of interest it is possible to achieve a specific biotinylation of the desired protein in vivo
with the E. coli biotin ligase BirA. BirA covalently attaches a single biotin to the lysine residue
of the avidin tag. The femtomolar affinity constant between biotin and streptavidin can be ex-
ploited for further purposes, purification attempts example.82 In previous works of the research
group Hellmich, an avidin tag was inserted on two different positions in hsV4N and hsV4N
∆N132: either before the C-terminal His6-tag (hsV4N Avi2) or after the C-terminal His6-tag
(hsV4N Avi3).76 For biotinylation of the avidin tagged proteins, E. coli BL21 (DE3) Gold cells
were heat-shock co-transformed with the respective plasmids, one expressing the BirA ligase
(p155) and the other the respective TRPV4 N-terminal construct. Cell cultivation and protein ex-
pression were similar as described in section 3.7.1. Additionally, 40 µM biotin were added to the
growth medium to ensure sufficient biotinylation of the target protein.82
Purification of biotinylated human N-terminal TRPV4 constructs
The same purification protocol as described in subsection 3.7.2 above was used with minor
deviations. The last wash step (W3) was left out, furthermore 6 elution steps with Ni2+-NTA
buffer 500 were performed. SEC were executed with a ENrich™ SEC 70 column. Purity was
analyzed via SDS-PAGE and Coomassie staining. Successful biotinylation was determined
during the purification via dot-blot with a streptavidin-POD (Strep-POD) antibody and also a
western blot with the same Strep-POD antibody was performed to verify the correct molecular
weight as described previously.76,77
3.7.5 Recombinant expression and purification of human RhoA
The same expression protocol as described in section 3.7.1 was used. Protein expression was
induced at an OD600 of 0.8. Table 3.11 lists the further expression conditions used for human
RhoA in this work.
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Chapter 3. Methods
Table 3.11: Expression conditions of human RhoA. IPTG conc. = IPTG concentration, Temp. =
Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
human RhoA-His6 LB 250 18 16
The same purification protocol as in subsection 3.7.2 was used with buffer conditions suited to
human RhoA (see also table 2.4). Furthermore, if needed, a TEV-cleavage was performed over
night to remove the n-terminal His6-tag (see also table 2.4 for buffer conditions) with subsequent
reverse IMAC. Reverse IMAC washing steps were performed until the OD280 dropped below 0.4.
Concentrated protein containing fractions were then subjected to SEC.
3.7.6 Recombinant expression and purification of human DDX3X_aa122-582
(DDX3X)
The same expression protocol as described in section 3.7.1 was used, but here the used an-
tibiotic was Kanamycin with end concentrations of 50 µg/mL. Protein expression was induced
at an OD600 of 0.8. Table 3.12 lists the further expression conditions used for hsDDX3X in this
work.
Table 3.12: Expression conditions of hsDDX3X. IPTG conc. = IPTG concentration, Temp. =
Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
human DDX3X_aa122-582-His6 LB 1000 18 25
The same purification protocol as in subsection 3.7.2 was used with buffer conditions suited to
hsDDX3X (see also table 2.5). Furthermore, the IMAC was differently performed as described
73
Chapter 3. Methods
in the following. The obtained supernatant after cell lysis and centrifguation of 1 L E. coli cell
culture was loaded onto 3 mL Ni-2+-NTA agarose provided in a column which was beforehand
equilibrated with 2 CV IMAC Wash A buffer. After discarding the flow-through the Ni-2+-NTA
agarose was washed with 15 mL IMAC wash buffer A, followed by 10 mL IMAC High salt buffer
and 10 mL IMAC Wash B buffer. Elution steps were performed with IMAC Elution buffer until the
OD280 dropped below 0.3. Furthermore, if needed, a TEV-cleavage was performed over night
to remove the n-terminal His6-tag (see also table 2.5 for buffer conditions) with subsequent
reverse IMAC. Reverse IMAC washing steps were performed until the OD280 dropped below 0.2.
Concentrated protein containing fractions were then subjected to SEC.
3.7.7 Recombinant expression and purification of human ITCH
The same expression protocol as described in section 3.7.1 was used. Protein expression in
the cells were induced at an OD600 of 0.8. Table 3.13 lists the further expression conditions
used for human ITCH in this work.
Table 3.13: Expression conditions of human ITCH. IPTG conc. = IPTG concentration, Temp. =
Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
human ITCH-nHis6 LB 250 18 16
The same purification protocol as in subsection 3.7.2 was used with buffer conditions suited to
human ITCH (see also table 2.6) with the minor deviaton during the IMAC, where three times
twice the CV was eluted. Furthermore, if needed, a TEV-cleavage was performed over night
to remove the n-terminal His6-tag (see also table 2.6 for buffer conditions) with subsequent
reverse IMAC. Reverse IMAC washing steps were performed until the OD280 dropped below 0.4.
Concentrated protein containing fractions were then subjected to SEC.
74
Chapter 3. Methods
3.7.8 Recombinant expression and purification of human ITCH WW domains
The same expression protocol as described in section 3.7.1 was used. Protein expression in
the cells were induced at an OD600 of 0.8. Table 3.14 lists the further expression conditions
used for human ITCH WW domains in this work.
Table 3.14: Expression conditions of human ITCH WW domains. IPTG conc. = IPTG concen-
tration, Temp. = Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
hsITCH_WW1 domain-His6 LB 250 20 16
hsITCH_WW2 domain-His6 LB 250 20 16
hsITCH_WW3 domain-His6 LB 250 20 16
hsITCH_WW4 domain-His6 LB 250 20 16
hsITCH_WW1+WW2 domains-His6 LB 250 20 16
hsITCH_WW3+WW4 domains-His6 LB 250 20 16
The same purification protocol as in subsection 3.7.2 was used with buffer conditions suited to
human ITCH WW domains (see also table 2.7), with just three elution steps during the IMAC.
Furthermore, if needed, a TEV-cleavage was performed over night to remove the n-terminal
His6-tag (see also table 2.6 for buffer conditions) with subsequent reverse IMAC. Reverse IMAC
washing steps were performed until the OD280 dropped below 0.2. Concentrated protein con-
taining fractions were then subjected to SEC.
3.7.9 Recombinant expression and purification of human YAP-WW domains
The same expression protocol as described in section 3.7.1 was used. Protein expression in
the cells were induced at an OD600 of 0.8. Table 3.15 lists the further expression conditions
used for human YAP WW domains in this work.
75
Chapter 3. Methods
Table 3.15: Expression conditions of human YAP WW domains. IPTG conc. = IPTG concentra-
tion, Temp. = Temperature
Construct Medium IPTG conc. [µM] Duration [h] Temp. [°C]
hsYAP1_WW1 domain-His6 LB 250 16 20
hsYAP1_WW2 domain-His6 LB 250 16 20
hsYAP1_WW1+WW2 domains-His6 LB 250 16 20
The same purification protocol as in subsection 3.7.2 was used with buffer conditions suited to
human YAP WW domains (see also table 2.9), with just three elution steps during the IMAC.
Furthermore, if needed, a TEV-cleavage was performed over night to remove the n-terminal
His6-tag (see also table 2.6 for buffer conditions) with subsequent reverse IMAC. Reverse IMAC
washing steps were performed until the OD280 dropped below 0.2. Concentrated protein con-
taining fractions were then subjected to SEC.
3.8 Pulldown assay of biotinylated hsV4N constructs
All steps of the below mentioned pulldown assay were carried out at 4 °C. HEK293 cells of a
10 cm dish were cultivated until 90 % confluency. After discarding the supernatant, cells were
washed once with 5 mL ice-cold PBS and incubated for 10 min in pulldown lysis buffer (see table
2.2) under mild agitation. Due to the short incubation in the digitonin-containing pulldown lysis
buffer, the plasma membrane of the HEK293 cells permeabilizes, allowing cytosolic proteins
to diffuse into the supernatant of the cells without destroying other cell compartments.83 The
supernatant of the still adherent cells was then transferred into a fresh tube and centrifuged for
10 min at 400 rpm. The supernatant again was transferred into a fresh tube and centrifuged for
30 sec at 12 000 rpm. The supernatant was transferred into a fresh tube and the protein con-
centration was determined via a Bradford Assay (see also sec 3.1.9). 20 µL streptavidin-coated
magnetic beads were pre-incubated with 500 ng the respective non- or biotinylated N-terminal
TRPV4 protein for 30 min at 4 °C under mild rotation. After three washing steps with PBS, the
pre-loaded beads were incubated in 250 µg of the digitonin-extracted HEK293 cytosolic fraction.
76
Chapter 3. Methods
To mimic an activation of the TRPV4 cation channel function, some samples were supplemented
with 2 mM CaCl2. After an incubation for 30 min at 4 °C under mild rotation, beads were washed
again three times with PBS and flash-frozen in liquid N2 until further use.
3.9 Mass spectrometry
Mass spectrometry based proteomics became an indispensable tool for the analysis of proteins
and their interaction partners. Several methods have been developed to investigate the complex
networks of cell proteomes. In cooperation with Dr. Ute Distler (AG Tenzer, FZI Core Facility for
Mass Spectrometry, Mainz) it was possible to approach towards an investigation of the human
TRPV4 N-terminus interactome via ultra high-definition mass spectrometryE (UDMSE). There
are several methods to prepare samples for mass spectrometry. In this work, samples were
obtained either after in-gel digestion (see subsection 3.9.1) or in-solution digestion (see sub-
section 3.9.2) after pulldown experiments with magnetic streptavidin beads (see section 3.8).
Digestions (see sections 3.9.1 and 3.9.2) were kindly performed by Ruben Spohrer (AG Tenzer,
FZI Core Facility for Mass Spectrometry, Mainz), MS measurements and data processing by Dr.
Ute Distler (AG Tenzer, FZI Core Facility for Mass Spectrometry, Mainz).
3.9.1 In gel digestion
After SDS-PAGE and Coomassie staining, the protein lanes of interest were cut out of the gel
and added to 200 µL 50 mM NH4HCO3 buffer in 50 % acetonitrile (ACN), followed by incubation
in an ultrasonic bath for 5 min. The supernatant was discarded and the foregoing step was
repeated. 200 µL 100 % ACN were added to the left gel pieces and again incubated in an ul-
trasonic bath for 5 min. The supernatant was discarded again and 100 µL 1 mM DTT solution
were added to the gel pieces, followed by an incubation for 60 min at 56 °C. After cooling down
the sample to RT, 100 µL iodoacteamide (IAA,c= 10 mg/mL) were applied and incubated for
45 min under light exclusion, followed by another addition of 100 µL 50 mM NH4HCO3 buffer
in 50 % acetonitrile (ACN) and incubation in an ultrasonic bath for 5 min. After discarding the
supernatant the last step was repeated. Afterwards, the gel pieces were incubated in 100 µL
100 % ACN in an ultrasonic bath for 5 min. The supernatant was discarded, the gel pieces
heated at 56 °C for 1 min and then 25 µL trypsin solution (dilution 1:100) were added. Incuba-
tion was performed over night at 37 °C and the supernatant was added in an ice cooled tube
77
Chapter 3. Methods
in which all following supernatants were collected. Now, the remaining gel pieces were incu-
bated in 50 µL 50 % ACN solution with 1 % formic acid (FA) in an ultrasonic bath for 15 min. This
step was performed twice, each time the supernatant was collected in the before mentioned
tube. 100 µL 100 % ACN were then added to the gel sample and again incubated in an ultra-
sonic bath for 15 min. The supernatant was collected again and the gel pieces were discarded,
followed by an incubation of the collected supernatants for 30 min at −80 °C. The collected
supernatants were then concentrated to a volume of 20 µL with a vacuum concentrator. and
then centrifuged at 13 000 rpm for 15 min at 4 °C. 15 µL of the supernatant were then added to
5 µL Enolase (100 fmol) and 5 µL 1 % FA. This mixture was then ready for mass spectrometric
measurements.
3.9.2 In-solution digestion
To obtain samples for mass spectrometric measurements after the pulldown assay described
in section 3.8, an in-solution digestion was performed. Proteins were precipitated with the "Pro-
teoExtract™Protein Precipitation Kit" (Merck Millipore, Darmstadt). 1 % RapiGest SF Surfactant
solution were added ( 110 of sample volume ) followed by an incubation for 15 min at 80 °C. 8 mM
DTT were added to the sample, incubated for 15 min at 56 °C, followed by the addition of 15 mM
IAA and an incubation at RT for 45 min. 1 µL trypsin and 8 mM DTT were added and incu-
bated over night at 37 °C . The pH of the sample was lowered to 3 with 1 M HCl an the sample
incubated for 10 min at 37 °C. After centrifugation for 30 min at 130 000 rpm and 4 °C, 10 µL su-
pernatant were mixed with 5 µL Enolase (100 fmol) and 10 µL 1 % FA to yield samples ready for
MS measurements.
3.9.3 Liquid Chromatography Mass Spectrometry
After tryptic digestion (see subsections 3.9.1 and 3.9.2) the peptides were separated via nanoU-
PLC with a nanoAcquity UPLC system (Waters Corporation, Milford) on an Acquity UPLC HSS-
T3 reverse phase column (high silica strength, 75 µm × 250 µm 1.8 µm), which was equilibrated
with Mobile Phase A (see table 2.2). In direct injection mode sample volumes between 0.7 µL
and 0.6 µL were applied onto the reverse phase column, followed by a gradient flow of 5-40 %
with Mobile Phase B over 90 min at a flow rate of 300 nL/min and a column temperature of
78
Chapter 3. Methods
55 °C. Afterwards the column was washed with 90 % of Mobile Phase B and furthermore re-
equilibrated.
A nano-ESI-Q-TOF mass spectrometer (Synapt G2-S HDMS, Waters Corporation) with an ion
mobility separation (IMS) device was used for MS analysis performed in positive mode ESI. LC-
MS data were collected in DIA mode using MSE combined with IMS (HDMSE) as well as in DDA
mode using a top 10 method selecting the 10 most abundant ions for fragmentation.63
3.9.4 Data Processing and Protein Identification
LC-MS DIA data were processed and searched with ProteinLynx Global SERVER (PLGS) (ver-
sion 3.02 built 5 from Waters Corporation). DDA data were processed using using the software
package PEAKS (version 8, Bioinformatic Solution Inc., Waterloo, Canada). Protein identifi-
cation was obtained searching a custom compiled database containing UniProtKB/SwissProt
entries of the E.coli and human reference proteomes (entries: 4434 E. coli, 20 231 human)
as well as the sequences of the N-terminal TRPV4 constructs. Sequence information for eno-
lase (Saccharomyces cerevisiae) and common contaminants (e.g. human keratins, BSA, and
trypsin) were added to the databases. Identified peptides had to match several search crite-
ria:
1. Trypsin had to be the digestive enzyme.
2. Up to two missed cleavages were allowed.
3. Carbamidomethyl cysteine was defined as a fixed modification.
4. Methionine oxidation was defined as a variable modification.
5. Peptides had to have a minimum length of six amino acids.
The false discovery rate (FDR) for peptide and protein identification was assessed searching a
reversed database. FDR was set to a 1 % threshold for database search in PLGS and PEAKS.
HDMSE collected data were post-processed with the in-house software ISOQuant in order to
facilitate the data analysis.63
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Chapter 3. Methods
3.10 Enzymatic assays
To validate the enzymatic activity of the recombinantly expressed and purified enzymes in the
course of this work, enzymatic assays were performed as described below.
3.10.1 GTPase assay
Intrinsic GTPase activity of recombinantly expressed and purified human RhoA (see also sub-
section 3.7.5) was determined via the GTPase-Glo Assay (see also table 2.18) according to
the manufacturers protocol. Luminescence was recorded with a FLUOStar Omega Microplate
Reader (BMG Labtech). In this assay, GTPase activity is inversely correlated to the measured
amount of luminescence.
3.10.2 ATPase assay
ATPase activity of recombinantly expressed and purified hsDDX3X (see als subsec3.7.6) was
determined via an cuvette-based in vitro-ATPase assay. The ATPase assay used in this work
is based on the enzyme-linked regeneration of ADP to ATP coupled to the oxidation of NADH
to NAD2.The decrease of NADH and therefore the ATPase dependent hydrolysis of ATP can
be determined with photometric measurements at 340 nm.84 For this, the components of ta-
ble 3.16 were mixed together in a cuvette with HEPES buffer (pH = 7). After 30 s 1 mM ATP
was added to the mixture and due to the ATPase-dependent RNA-Helicase Activity of DDX3X,
the mixture was supplemented with 1 µM palindromic duplex RNA after 5 min ′(Sequence: 5 -
3′UUUUUUUUUUUUUUUUUUUUGGCGGCCGCC- ). As a control, the well-characterized B.
subtilis ATPase BmrA was also supplemented with hsV4 ARD to rule out unspecific effects on
the lactate dehydrogenase and pyruvate kinase involved in this assay. Measurements were
carried out at RT with a Perkin-Elmer Lambda 25 Photometer.
Table 3.16: Sample composition for the in vitro-ATPase assay
Component
MgCl2 1 µM
Ascorbinic acid 1 µM
80
Chapter 3. Methods
Component
Phosphoenolpyruvate 3 µM
Pyruvate kinase 0.05-0.08 units/µL
Lactate dehydrogenase 0.08-0.12 units/µL
hsDDX3X 3 µM
BmrA 3 µM
hsV4N construct 12 µM
duplex RNA 1 µM
ATP 1 µM
3.10.3 Ubiquitinylation assay
To determine the E3 Ubiquitin ligase activity of recombinantly expressed and purified human
ITCH (see also subsection 3.7.7) an in vitro-ubiquitinylation assay was performed. For this,
the components of table 3.17 were incubated at 30 °C for up to 3 h in 10 mM Tris (pH=7.4) with
5 mM MgCl2. Out of this mixture, samples were taken after several time points for subsequent
SDS-PAGE and Coomassie staining.
Table 3.17: Sample composition for the in vitro-Ubiquitinylation assay
Component Concentration [µM]
ATP 5000
Ubiquitin 100
E1 Ubiquitin ligase UBE1 2
E2 Ubiquitin ligase Ubc5Hc 1
E3 Ubiquitin ligase ITCH 10
hsV4N construct 20
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Chapter 3. Methods
3.11 Blue Native-PAGE (BN-PAGE)
BN-PAGE is a straight-forward way to identify direct protein-protein interactions, especially for
proteins which isoelectrical points are > 8, as these proteins do not migrate in a PAGE under
Laemmli-conditions.85 Here, Coomassie G-250 is added into the discontinuous PAGE to create
a net negative charge to allow protein migration towards the anode. In this work, BN-PAGE
was used to determine the minimal binding side of ITCH at hsV4N. For this, 10 µM ITCH and
20 µM of the respective hsV4N construct were mixed in a tube with 10 mM Tris (pH=7.4) sup-
plemented with 5 mM MgCl2 as reaction buffer. The protein mixture was incubated at 30 °C for
1 h. After this, the protein mixture was supplemented with Coomassie G-250 to an end concen-
tration 0.25 % (w/v). The PAGE were run with 4 - 15 % Mini-Protean® TGX™ Precast Protein
Gels with a electrophoresis cathode buffer (see table 2.2) for 3 h at 4 °C.Ater this, Coomassie
staining was performed and relevant bands were cut out to provide them for mass spectromet-
ric verification (Mass Spectrometry Core Facility, Johns Hopkins School of Medicine, Baltimore,
USA).
3.12 Circular dichroism (CD) spectroscopy
Circular dichroism (CD) measurements were performed on a Jasco-815 CD spectrometer with
a spectral range between 195 and 260 nm at 20 °C. The scanning intervals were set to 1 nm,
the scanning speed to 50 nm/min and the band-width to 5 nm. CD data are often reported as a
mean residue ellipticity [Θ] 2 -1mrw,λ (deg cm dmol ). The conversion from the measured ellipticity
Θ (deg) to the mean residue ellipticity [Θ]mrw,λ is described by equation 3.1.
[Θ]mrw,λ =
MRW · [Θ]
10 (3.1)· c · d
Mean residue weight (MRW) is calculated with MRW = MN−1 , were M is the molecular weight
in Dalton and N is the amino acid number of the measured protein. The secondary structure
content was predicted with analyzing the CD spectra with the CAPITO tool (https://capito.
uni-jena.de).The Chou-Fassmann algorithm predicts secondary structures based on empiric
data in which amino acids show preferences for distinct secondary structures. Furthermore,
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Chapter 3. Methods
CAPITO compares the measured spectra with basis spectra data sets. A nearest-neighbor
approach determines the 25 best matching references curves defined by closest proximity. At
last, the lowest area differences are compared to reference curves.
3.13 NMR spectroscopy
To obtain uniformly 15N-labeled proteins for 15N-HSQC spectra, E. coli BL21 Gold cells were
transfected and the proteins expressed as described in sections 3.1.1 and 3.7, however, the
cells were grown in M9 medium (see table 2.2) to ensure that 15NH4Cl is the only nitrogen
source for protein expression. NMR spectra of respective proteins were recorded at 25 °C on
Bruker AVANCE 600, 700 and 800 MHz spectrometers equipped with cryogenic triple reso-
nance probes (Bruker, Karlsruhe) at the Centre for Biomolecular Magnetic Resonance (BMRZ)
(Goethe-University, Frankfurt a.M.). 1H,15N-HSQC spectra were measured using standard
pulse sequences and processed using TOPSPIN 3.5 (Bruker, Karlsruhe).86
3.14 Cross-linking mass spectrometry (XL-MS)
XL-MS is a powerful tool for investigations of protein-protein interactions (PPIs) and the inter-
faces of these interaction. Due to the chemical cross-linking between functional groups of amino
acid side chains XL-MS is advantageous to capture and identify weak or transient PPIs. Here,
H12/D12-disuccinimidyl suberate (H12/D12-DSS) was used, cross-linking between the amide
groups of lysines. Purified (hsV4N and DDX3X as well as hsV4N and ITCH) proteins were mixed
in a molar ratio of 1:1 to a final concentration of 1 mg/mL in 100 µL. H12/D12-DSS was freshly
dissolved in DMF prior use and added to the protein mixture at a final concentration of 1.5 mM.
The cross-linking reaction was incubated for 2 h on ice and quenched with the addition of am-
monium bicarbonate at a final concentration of 500 mM. The mixture was further incubated on
ice for additional 2 h. Samples were flash-frozen in liquid nitrogen and stored at -20 °C until
further use. Sample processing, MS measurements and analysis were kindly carried out by M.
Sc. Jasmin Jansen (Working group Stengel, University of Konstanz).87
83
Chapter 4. Results
4 Results
4.1 A beginning to elucidate the versatile TRPV4 N-terminal
protein interactome - the tip of the iceberg
4.1.1 Purification of biotinylated TRPV4 N-terminal proteins
The protein interactome of a cell is not a static structure of probable binary protein-protein in-
teractions (PPIs). It is a reflection of the cells state and therefore indicates protein abundance, 
post-translational modifications, cell cycle phase or the stress level of a cell. Due to this broad 
variety of possible conditions, the protein interactome of a cell is very dynamic and specific 
biochemical methods can only yield snapshots of protein interactions in certain biological situa-
tions. To obtain a comprehensive view of the protein interactome, shotgun mass spectrometry 
is the method of choice.73,88 This work focuses on the protein interactome of the full-length 
TRPV4 N-terminus (here referred to as hsV4N), as this portion of the full-length ion channel is 
a notorious hot-spot for disease-causing point mutations.27,56,89,90 Furthermore, hsV4N plays 
an integral role in channel sensitization as well as desensitization and harbors a number of 
pivotal protein, lipid and small molecule interaction sites, like a phosphoinositide binding do-
main (PBD) and a proline rich region (PRR), which are followed by six α-helical ankyrin re-
peats, the ankyrin repeat domain (ARD).22,39 Previously, a protocol was established to obtain 
the hsV4N protein interactome after pulldown assays in HEK293 cell lysates followed by shot-
gun proteomics performed in cooperation with Dr.  (RG  University Medicine Mainz, 
JGU Mainz).76 This protocol required recombinant expression, in vivo biotinylation as well as 
protein integrity investigations of several TRPV4 N-terminal constructs with an so-called avidin-
tag (here referred as Avi). This tag allows side-specific in vivo biotinylation of a lysine in the 
avidin-tag itself (single letter code avidin-tag: GLNDIFEAQKIEWHE).91 In addition to
84
Chapter 4. Results
the already used and validated full-length hsV4N with avidin-tag (hsV4N wt Avi), before men-
tioned protocol was optimized for the TRPV4 ankyrin repeat domain (here referred as hsV4
ARD), including the selected disease-causing mutations R232C and K276E (figure 4.1). The
TRPV4 R232C mutation leads to Charcot-Marie-Tooth Type 2C (CMT2C)54, whereas TRPV4
K276E results in fetal akinesia - a combined phenotype of metatropic dysplasia with putative
neurodegenerative symptoms.92 Protein expression, in vivo biotinylation and purifications were
performed as described in subsection 3.7.4. All size exclusion chromatograms (SEC) of used
TRPV4 N-terminal constructs in figure 4.1 show narrow gaussian peaks with expected elu-
tion volumes for all biotinylated proteins (see section 6.2 for SEC purification of unbiotinylated
proteins). Coomassie stained SDS-PAGEs show pure proteins and a Streptavidin-Peroxidase
(Strep-POD) dot-blot confirms successful biotinylation. However, hsV4N wt Avi shows a sig-
nificant stronger signal in the Strep-POD dot blot than the hsV4 ARD Avi constructs. Image
analysis of the Strep-POD dot blots with Fiji shows a 90 % weaker biotinylation efficacy of the
hsV4 ARD Avi constructs compared to hsV4N Avi (data not shown). Adjustment of the used
hsV4 ARD Avi concentrations was sufficient enough for successful pulldowns due to the strong
interaction between biotin and streptavidin with a K in the femtomolar range.91,94–96D CD spec-
tra show, as also previously reported22, that hsV4N consists of a large α-helical part, which is
contributed by the ARD (aa 149 - aa 398), and a large unstructured region (aa 1-aa 149). Com-
parison of the CD spectra between biotinylated and non-biotinylated hsV4 ARD Avi constructs
shows no change in the α-helical structure, which is also underlined by secondary structure pre-
diction via CAPITO93. Only biotinylated hsV4N wt Avi supposedly harbors a marginal β-sheet
Figure 4.1 (facing page): Purification of in vivo biotinylated and structural intact TRPV4 N-terminal constructs. A Schematic
topology model of TRPV4 N-terminal constructs used for protein interactome investigations via pulldown assays and subsequent
mass spectrometry in this work. HsV4N represents the full-length human TRPV4 N-terminus with important interaction sites like the
phosphoinositide binding site (PBD), proline rich region (PRR) and the ankyrin repeat domain (ARD). Avi = avidin tag (single letter
code: GLNDIFEAQKIEWHE). His6 = hexa-histidine tag (single letter code: HHHHHH). B SEC purifications of biotinylated hsV4N
wt Avi and hsV4 ARD Avi constructs. SEC runs were either performed with a HiLoad Superdex 200 pg or a HiLoad Superdex
75 pg preparative SEC column. Inlets show Coomassie stained 15 % SDS-PAGEs of collected and concentrated fractions after
SEC, furthermore a Strep-POD dot blot indicating biotinylated proteins of the respective proteins. x-axis: eluted volume in mL,
y-axis: absorbance at 280 nm, M = marker in kilodalton (kDa). C Comparison of far UV CD spectra of purified biotinylated and
non-biotiynlated hsV4N constructs. Spectra were measured at 293 K and proteins were used at 2µM concentration in SEC buffer
with a final concentration of 30 mM NaCl. Inlets show secondary structure prediction performed with CAPITO.93 The predicted
α-helix/β-sheet/random coil content of the purified proteins is for non-biotinylated hsV4N wt Avi 53 %/0 %/46 %, hsV4 ARD wt
Avi 76 %/0 %/24 %, hsV4 ARD R232C Avi 74 %/0 %/26 % and hsV4 K276E Avi 76 %/0 %/24 %. Biotinylated proteins show the
following secondary structure content distribution: hsV4N wt Avi 40 %/14 %/46 %, hsV4 ARD wt Avi 76 %/0 %/24 %, hsV4 ARD
R232C Avi 74 %/0 %/26 % and hsV4 ARD K276E Avi 76 %/0 %/24 %. x-axis: mean residue ellipticity ([Θ] −3 2mrw,λ) in 10 deg cm
dmol−1, y-axis: wavelength λ in nm.
85
Chapter 4. Results
A 149 398
hsV4N wt Avi N PBD PRR ARD Avi His C6
hsV4 ARD wt Avi N ARD Avi His C6
R232C
hsV4 ARD R232C Avi N ARD Avi His C6
K276E
hsV4 ARD K276E Avi N ARD Avi His C6
B
350 hsV4N wt Avi biotinylated 150 hsV4 ARD wt Avi
hsV4 ARD R232C Avi
hsV4 ARD K276E Avi
300
M biotinylated M
[kDa] Coomassie Strep-POD [kDa] Coomassie Strep-POD
250
100
wt
200 50 50
37 37 R232C
150
25
25 wt C
50 E32 6 K276E2
R 2
7
100 K
50
Superdex 200 Superdex 75
0 0
40 50 60 70 80 90 100 40 50 60 70 80 90 100
Volume [mL] Volume [mL]
C
1 .0 1 .0
random coil
60 hsV4N wt Avi 60 hsV4N wt Avi
random coil
0 .9 b-sheet 0 .9 b-sheet
hsV4 ARD wt Avi a-helix hsV4 ARD wt Avi a-helix
0 .8 0 .8
hsV4 ARD R232C Avi hsV4 ARD R232C Avi
0 .7 0 .7
hsV4 ARD K276E Avi hsV4 ARD K276E Avi
0 .6 0 .6
40 biotinylated 40
0 .5 0 .5
0 .4 0 .4
0 .3 0 .3
0 .2 0 .2
20 20
0 .1 0 .1
0 .0 0 .0
N wt D D t
V4 D
 R 2C R 6E 4
N  w RD C RD EA
hs R 3
A 7
A s
V RD A 32 A 76
R2 K2 h A R2 K2
0 0
-20 -20
-40 -40
200 210 220 230 240 250 260 200 210 220 230 240 250 260
l [nm] l [nm]
86
Abs [mAu]
2 -1 280nm-3
q [10 deg cm dmol ]
mrw, l
sec. structure content
-3 2 -1
Abs [mAu]
280nm
q [10 deg cm dmol ]
mrw, l
sec. structure content
Chapter 4. Results
fold content compared to non-biotinylated hsV4N wt Avi.
4.1.2 The TRPV4 N-terminal protein interactome in HEK293 cells derived via
mass spectrometry
For the elucidation of the TRPV4 N-terminal protein interactome, biotinylated and non-biotinylated
constructs (see subsection 4.1.1) were subjected to streptavidin-dependent pulldowns with
HEK293 cytosolic extracts. Table 4.1 lists all samples which were used in the pulldowns with
HEK293 cytosolic protein extracts (see figure 4.2 for workflow and section 3.8 for protocol).
Shortly, streptavidin-coated magnetic beads were incubated with the respective purified TRPV4
N-terminal construct (biotinylated or non-biotinylated), washed and then incubated with the cy-
tosolic extract of HEK293 cells. Additionally, samples with biotinylated constructs were sup-
plemented with 2 mM Ca2+ during the HEK293 cytosolic extract incubation step to mimic an
increased intracellular Ca2+ concentration as it would be observed upon TRPV4 ion channel
activation.
Table 4.1: Sample overview of hsV4N interactome pulldowns with HEK293 cytosolic protein
extracts
Construct Biotinylated Sample supplement
hsV4N wt Avi Yes none
hsV4 ARD wt Avi Yes none
hsV4 ARD R232C Avi Yes none
hsV4 ARD K276E Avi Yes none
hsV4N wt Avi Yes 2 mM Ca2+
hsV4 ARD wt Avi Yes 2 mM Ca2+
hsV4 ARD R232C Avi Yes 2 mM Ca2+
hsV4 ARD K276E Avi Yes 2 mM Ca2+
Controls
hsV4N wt Avi No none
hsV4 ARD wt Avi No none
hsV4 ARD R232C Avi No none
hsV4 ARD K276E Avi No none
87
Chapter 4. Results
Extracting cytosolic proteins Streptavidin-Biotin Pulldown Shotgun proteomics
A HEK293 cells B C putative
interactors
interactor trypsin
digestion
Streptavidin bait
tetramer (e.g. hsV4N wt Avi) peptides
Digitonin
extraction
UPLC +
= biotin ESI
E
back- UDMS +
ground DIA
= putative magnetic
interactor bead m/z
Figure 4.2: Outline of the proteomics workflow performed in this work. See sections 3.8 and 3.9 for protocols. A To enrich
cytosolic proteins, plasma membranes of HEK293 cells were permebealized with digitonin. This detergent releases cytosolic
proteins into the supernatant while contaminations by other cell compartments are reduced to a minimum.83 B Schematic layout
of the hsV4N interactome pulldown. Streptavidin-bound magnetic beads were loaded with respective purified hsV4N Avi construct
(see also figure 4.1). Loaded beads were incubated in HEK293 protein extract containing cytosolic proteins. As a control, beads
were also incubated with non-biotinylated TRPV4 N-terminal constructs to rule out unspecific interactors (background).C After
elution, putative interactors were trypsin digested to peptides. These peptides were then separated by ultra high-performance
liquid chromatography (UPLC) and directly supplied into the mass spectrometer via electron spray ionization (ESI). MS data were
collected in data independent acquisition (DIA) mode. All steps in C were performed in cooperation with
University Medicine Mainz).63,72,73
The HEK293 protein interactome of the TRPV4 full-length N-terminus
Figure 4.3 shows the volcano plots of two independent ultra high-definition mass spectrometryE
(UDMSE) measurements of hsV4N wt Avi. In these volcano plots, only significantly detected
proteins are shown. All samples listed in table 4.1 were measured in technical quadruplicates.
Proteins were considered as significant, if the p-value was ≤ 0.05 (Benjamini-Hochberg cor-
rected Student’s t-test) and the log2 fold change fo respective protein between biotinylated
hsV4N wt Avi and non-biotinylated hsV4N Avi wt control sample was ≥ 1. Additionally, only pro-
teins which appeared in both independent UDMSE measurements were considered as highly
probable hsV4N wt Avi protein interactome members. Significant proteins showed different fold
changes between the two independent measurements (black and grey dots in figure 4.3), in-
dicating a high sensitivity of the UDMSE measurements towards different NaCl concentrations
88
Chapter 4. Results
A B
12 12 2+
+ 2 mM Ca
11 11
10 10
9 hsV4N wt Avi 9
8 8 hsV4N wt Avi
7 hsV4N wt Avi 7
6 6
5 5
4 4 hsV4N wt Avi
3 3
2 2
1 1
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
log2 fold change (biotinylated/non-biotinylated) log2 fold change (biotinylated/non-biotinylated)
Figure 4.3: UDMSE measurements revealed up to 64 probable hsV4N protein interactors. A Volcano plot of two independent
HEK293 protein interactome UDMSE measurements from hsV4N wt Avi. 34 proteins were considered as significant. B Volcano
plot of two independent HEK293 protein interactome UDMSE measurements from hsV4N wt Avi with 2 mM Ca2+ sample supple-
mentation. 64 proteins were considered as significant. Black (hsV4N wt Avi, 300 mM NaCl buffer) and dark gray points (hsV4N wt
Avi, 750 mM NaCl buffer) indicate significant, detected HEK293 protein interactome candidates in both independent experiments.
Each pulldown was measured in quadruplicates. Horizontal light gray lines indicate the p-value = 0.05 (Benjamini-Hochberg cor-
rected Student’s t-test) of the quadruplicates of each UDMSE measurement. Vertical light gray lines indicate a log2 fold change ≥
1. For a full list of hsV4N interactors, see tables 6.3 and 6.4
supplemented in the hsV4N wt Avi SEC buffer. Data analysis resulted in 34 relevant hsV4N wt
Avi interaction partners in samples without Ca2+ supplementation and 64 hsV4N wt Avi interac-
tion partners in the samples with Ca2+ supplementation (see figures 4.4 A and 4.5 A), including
the known direct TRPV4 interactor PACSIN3 in both conditions.22,23,70,71 Gene Ontology enrich-
ment analysis (GO enrichment analysis) is a technique to interpret high-throughput data in terms
of over- or underrepresentation of certain genes in respective experiment. The Gene Ontology
classification assigns genes to sets of predefined bins depending on their role in cellular pro-
cesses or functional characteristics.97 GO enrichment analysis with STRING98 via Cytoscape
3.8.0 demonstrated that all detected proteins are associated with the cytoplasm, which is com-
posed out of the cytosol and cytoskeleton, indicating successful extraction of mainly cytosolic
proteins. Only HNRNPM_HUMAN represents an exception, as it is associated with the nucleo-
plasm but also with ribonucleoprotein complexes - like the majority of the cytosolic proteins in the
here disclosed network. Like HNRNPM_HUMAN, a small portion of other proteins could also
be associated with the nucleoplasm, but are known for their nucleocytoplasmic shuttling.99 Ad-
ditionally, GO enrichment analysis and manual data curation connected a considerable amount
of detected proteins with cytoplasmic ribonucleoprotein or cytoplasmic stress granules (13 pro-
teins, see figure 4.4 and table 6.3). Cytoplasmic ribonucleoprotein granule is a collective GO
term for distinct RNA-binding protein-containing foci in the cytoplasm, including amongst others
neuronal transport granules, P-bodies and stress granules. Stress granules, appearing as their
name says after various stress factors like oxidative or hyperosmotic stress in the cytosol, form
89
-log10 (p-value)
-log10 (p-value)
Chapter 4. Results
via liquid-liquid phase separation of pre-existing RNA-binding protein (RBP) complexes.100,101
The exact role of stress granules in neuronal (patho)physiological processes remains largely
unknown. It is known that stress granule formation is accompanied by polysome disassembly
and translational arrest.102–105 Stress granules are mostly composed out of translation initia-
tors, RBPs, 40S ribosomes and polyadenylated mRNAs and therefore are proposed to play a
protective role in cells by promptly providing the undamaged translation machinery for important
household proteins after their disassembly due to stress withdrawal.106–110 Stress granules and
other cytoplasmic ribonucleoprotein granules show major overlaps in their proteome but are
also known for their highly variable and dynamic protein compositions, transitioning from one
granule to the other in dependency of the cellular stress level.101,107–109,111–114 Liu-Yesucevitz
et al. showed that potassium chloride (KCl)-induced depolarization in primary neurons, leading
to dramatically elevated cytoplasmic Ca2+ levels, induces a transformation of neuronal mRNA
transport granules to stress granules.115 Interestingly, it was shown that neuropathy-causing
mutations of TRPV4 show an elevated baseline Ca2+-influx activity of the channel, also leading
to higher cytosolic Ca2+-concentrations.53,54 In the present work, upon Ca2+-supplementation,
the amount of ribosomal proteins in the hsV4N interactome considerably increased, but also
the number of proteins associated with cytosolic ribonucleoprotein granules (20 proteins, figure
4.5 and table 6.4). In both conditions, without and with Ca2+, known stress granule formation
proteins are present, namely YBX1, IGF2BP1, PAPBC1 and DDX3X.107,108,116–120 Furthermore,
upon Ca2+ supplementation proteins associated with the GO term supramolecular fiber were
detected. Supramolecular fibers are defined as protein complexes which form fiber-shaped
structures, like microtubule or actin filaments. Fiber-like structures were shown to spur the mat-
uration of phase-seperated droplets to more stable and less dynamic structures and therefore
impair an important property of stress granules: the fast disassembly within minutes after stress
removal.101,102,117,121–130 Amyotrophic lateral sclerosis (ALS)-causing mutations in stress gran-
ule proteins, especially in the intrinsically disordered regions (IDRs) of RBPs, were shown in
cellulo to lead to insoluble inclusions. This is particularly interesting, as the TRPV4-associated
neuropathy Charcot-Marie-Tooth Type 2C is also referred as a so-called "ALS-mimic", due to the
shared clinical symptoms.131 Furthermore, insoluble inclusion formations were also observed
Figure 4.4 (facing page): The hsV4N protein interactome determined via UDMSE. A STRING interaction network of detected,
significant hsV4N interactome proteins. Outer ring color of nodes represent Gene Ontology (GO) terms for cell components, also
shown in B. For a full list of protein interactors see table 6.3 B Selected GO terms for cell components of significant, detected
hsV4N interactome proteins via UDMSE after GO enrichment analysis. C Selected GO terms for cell processes of significantly
detected hsV4N interactome proteins via UDMSE after GO enrichment analysis (Benjamini-Hochberg corrected, p<0.05)
90
Chapter 4. Results
RNP granules/
A stress granules
B
ribonucleoprotein
complex
ribosome
cytoplasmic ribonucleo-
protein granule
CRD-mediated mRNA
stability complex
nucleoplasm
cytoplasm
cytoplasmic
stress granule
0 5 10 15 20 25 30 35 40
C -log10 (p-value)
mRNA metabolic process
translational initiation
nuclear-transcribed mRNA
nonsense-mediated decay
nuclear-transcribed mRNA
catabolic process
gene expression
SRP-dependent cotranslational
protein targeting to membrane
protein localization to organelle
translation,
negative regulation of
peptide metabolic process
cellular protein localization
establishment of protein
localization to organelle
cellular localization
negative regulation of macro-
molecule metabolic process
macromolecule localization
RNA processing
0 5 10 15 20 25 30 35 40 45 50
-log10 (p-value)
91
Chapter 4. Results
with wild-typic RBPs after induction of repeated cycles of stress granule assembly and disas-
sembly in short time, mimicking chronic stress conditions.100,132 Permanent Ca2+ influx, for
example via neuropathy-causing mutations in TRPV4, could thus lead to a pathological switch
from dynamic granules to aggregates, which then impair the important roles of ribonucleopro-
tein granule functions like dendritic and axonal mRNA transport and local translation. Due to the
unique cellular structure of neurons, this cell type is more sensitive to the lacking clearance of
these aggregates and therefore explain the high neurotoxicity of these inclusions.133–135 Thus,
TRPV4 and its disease-causing mutations provide multiple factors to be a part of the stress
granule machinery. Neuropathy causing TRPV4 mutations, like TRPV4 R232C, lead to chronic
stress levels due to increased Ca2+-influx activity. Based on the data shown here, hsV4N itself
could act as a direct interaction site for stress granule key proteins and in combination with the
Ca2+-dependent recruitment of proteins involved in supramolecular fiber formation, possibly
leading to neurotoxic aggregates.
To narrow down the interaction sites of the previously identified hsV4N interactors, several hsV4
ARD constructs were probed in the same UDMSE experimental set-ups as hsV4N. The ARD
of TRPV4 is a hot-spot for disease-causing mutations.27,56 Here, besides the wild-typic hsV4
ARD, hsV4 ARD harboring the neuropathy-causing R232C mutation as well as the fetal-akinesia
causing hsV4 ARD K276E mutation were investigated with regard to their HEK293 cells protein
interactome. Figures 4.6 A-F show the STRING interaction map and GO enrichment analysis of
the respective hsV4 ARD construct without and with Ca2+-supplementation. To provide a better
overview, only proteins associated with ribonucleoprotein granules and/or stress granules via
GO analysis and/or manual data curation are shown in the interaction maps (figures 4.6 A-E).
Upon Ca2+-supplementation, the amount of RBPs increases in the case of hsV4 ARD wt (fig-
ures 4.6 A and B) and R232C (figures 4.6 C and D). Only for hsV4 ARD K276E the amount
of riboluceoprotein granule associated proteins decreases upon Ca2+-supplementation (figure
4.6 E and F). It should be noted that TRPV4 R232C showed elevated basal Ca2+-influx level in
cellulo.53,54 Basal Ca2+-influx activities of TRPV4 K276E were not evaluated until now, but due
to the severe clinical outcome of this mutation it could be assumed that this mutation also leads
to a gain-of-function phenotype of the channel.48 It is therefore possible that the interactions
after Ca2+ supplementation are more representative for the possible aberrant PPIs in the pres-
ence of disease-causing mutations (figures 4.6 C and D (hsV4 ARD R232C) as well as E and
F (hsV4 ARD K276E), respectively).
Comparing the interactome composition between the different TRPV4 N-terminal constructs, it
seems that the interactome is highly variable - not only between the samples without and with
92
Chapter 4. Results
Ca2+-supplementation, but also between hsV4 ARD wt and mutated hsV4 ARDs, as well as
between hsV4N and hsV4 ARD wt (see figure 4.7). These variations could thus hint towards
an important regulatory role of the intrinsically disordered region (hsV4 IDR), preceding hsV4
ARD in hsV4N, on the protein interactions of hsV4 ARD. Interestingly, throughout all UDMSE
measurements, the ATP-dependent RNA-helicase DDX3X was detected as a part of the hsV4N
protein interactome, more precisely of hsV4 ARD protein interactome. DDX3X was shown to
directly interact with full-length TRPV4 via a yeast-two hybrid screening (Y2H), although the
species of used TRPV4 was not mentioned in the publication.136 Due to the central role of
DDX3X in stress granule formation and its role in neurodegeneration, section 4.2 gives a closer
look on the probable direct interaction between DDX3X and hsV4N.119,137–139
The TRIP database (http://trpchannel.org/) is a manually curated database of PPIs for mam-
malian TRP channels and therefore gives an overview of known PPIs of TRPV4.16,29,30 Most
of the listed interactors in the TRIP database were determined via co-immunoprecipitations
(co-IPs), co-immunofluorescence staining and/or FRET measurements with endogenous pro-
teins and/or overexpressing HEK293 cells (see also table 6.1). The TRPV4 interacting protein
classes which are represented in the TRIP databsae are very diverse, including amongst others,
transmembrane proteins (TRPC1, AT1aR, AQP4 e.g.), kinases (FYN, LYN e.g.), E3 ubiquitin
ligases (ITCH) and cytoskeletal proteins (α-actin and α-tubulins). Another reliable database for
PPIs is BioGrid (https://thebiogrid.org/). BioGrid lists the same protein interactors as the TRIP
database for human TRPV4.140 In both databases, no RBPs or other proteins involved in cyto-
plasmic ribonucleoprotein granules are indicated. One reason for this could be that most of the
PPI determinations were conducted via co-IPs of full-length TRPV4 and subsequent western
blotting with immunostaining, only allowing to investigate pin-pointed PPIs with certain proteins.
Comprehensive mass spectrometric proteomics analysis of isolated TRPV4 domains like the
here used hsV4N as a bait were not published until now. Donate-Macian et al. used full-length
TRPV4 as a bait for Y2H screening of direct TRPV4 interactors, the species of the used TRPV4
construct was not mentioned though, as pointed out above. Besides DDX3X, UBAP2L was
also shown to directly interact with TRPV4.136 In this thesis, UBAP2L was detected in UDSME
Figure 4.5 (facing page): The hsV4N wt Avi protein interactome determined via UDMSE with Ca2+ sample supplementation.
A STRING interaction network of detected, significant hsV4N interactome proteins. Outer ring color of nodes represent Gene
Ontology (GO) terms for cell components, also shown in B. For a full list of protein interactors see table 6.4 B Selected GO
terms for cell components of significant, detected hsV4N wt Avi interactome proteins via UDMSE after GO enrichment analysis.
C Selected GO terms for cell processes of significant, detected hsV4N interactome proteins via UDMSE after GO enrichment
analysis.
93
Chapter 4. Results
RNP granules/
stress granules
A
B
ribonucleoprotein
complex
ribosome
cytoplasm
cytoplasmic ribonucleo-
protein granule
CRD-mediated mRNA
stability complex
cytoplasmic
stress granule
nucleoplasm
supramolecular fiber
0 5 10 15 20 25 30 35 40
C -log10 (p-value)
nuclear-transcribed mRNA
nonsense-mediated decay
SRP-dependent cotranslational
protein targeting to membrane
translational initiation
nuclear-transcribed
mRNA catabolic process
mRNA metabolic process
translation
nucleobase-containing compound
catabolic process
peptide metabolic process
protein localization to organelle
establishment of protein
localization to organelle
negative regulation
of gene expression
intracellular protein transport
cellular protein localization
negative regulation of macro-
molecule metabolic process
cellular localization
0 5 10 15 20 25 30 35 40 45 50
-log10 (p-value)
94
Chapter 4. Results
experiments conducted with hsV4 ARD wt (figures 4.6 A and B). However, the protein over-
lap between UDSME and Y2H is low, which was also already shown after comparison of other
affinity purification MS (AP-MS) experiments with Y2H screens with the same bait protein. This
emphasizes the highly complementary nature of both techniques: while MS approaches provide
large-scale data on protein complexes, with additional Y2H results it is possible to break these
complexes down into binary PPIs.141
2+
A B + 2 mM Ca
A
C R
A
D C R2 2 D
23 K
3
R A 2
2 K
v 7 R i A
27
D vi i 6
R A E
D v v 6
R A i E
A A
2 79 h 5 34s hV t s
t 4 V
w A N  
w 4
 
7 v  D i 5 A
N
D
R i 1 9 i
w R v 9 v  
v t A 6A i
w
t
A A
2 1 7 1
21 22 50 33
13 2 0 20 8
1
1 0 3 5
0 5
Figure 4.7: Venn diagrams of overlapping proteins detected in respective UDMSE experiments without (A) and with Ca2+
sample supplementation (B). For full lists of detected proteins see tables 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 6.10
Figure 4.6 (facing page): Interaction map, volcano plots and enrichment analysis of Gene Ontology (GO) cell component
and processes terms of hsV4 ARD wt Avi, hsV4 ARD R232C Avi and hsV4 ARD K276E Avi interactome proteins detected
via UDMSE without (A,C,E) and with Ca2+ sample supplementation (B,D,F) For full lists of protein interactors see tables 6.5,
6.6, 6.7, 6.8, 6.9 and 6.10 A hsV4 ARD wt Avi Upper left: STRING interaction network of detected, significant hsV4 ARD wt Avi
interactome proteins associated with cytoplasmic ribonucleoprotein granules and/or stress granules after manual data curation. For
full list of interactors see section 6.3. Outer ring color of nodes represent GO cell component terms shown in the GO cell component
enrichment graphic below. Upper right: Volcano plot of two independent HEK293 protein interactome UDMSE experiments with
hsV4 ARD wt Avi. Each pulldown was measured in quadruplicates. Horizontal light gray line indicates p-value = 0.05 (Benjamini-
Hochberg corrected Student’s t-test). Vertical light gray line indicates log2 fold change ≥ 1. Lower left: Selected GO terms for cell
components of significant, detected hsV4 ARD wt Avi interactome proteins via UDMSE after GO enrichment analysis (Benjamini-
Hochberg corrected, p<0.05). Lower Right: Top 15 GO cell processes terms of significant, detected hsV4 ARD wt Avi interactome
proteins via UDMSE after GO enrichment analysis (Benjamini-Hochberg corrected, p < 0.05). B hsV4 ARD wt Avi with Ca2+. C
hsV4 ARD R232C Avi and D with Ca2+. E hsV4 ARD K276 Avi and F with Ca2+. Please note for graphs in E and F the range
changing in the x-axis of the GO Enrichment Processes Graphs in comparison to A-D.
95
Chapter 4. Results
A
25
20
15
10
5
hsV4 ARD wt Avi
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
RNA binding
ribonucleoprotein
complex mRNA binding
mRNA 5’-UTR binding
cytoplasm N6-methyladenosine-
containing RNA binding
mRNA 3’-UTR binding
nucleoplasm structural constituent of ribosome
organic cyclic compound binding
heterocyclic compound binding
ubiquitin ligase inhibitor activity
5S rRNA binding
rRNA binding
Tat protein binding
G-rich strand telomeric DNA binding
structural molecule activity
ubiquitin protein ligase binding
0 5 10 15 20 25 0 5 10 15 20 25
-log10 (p-value) -log10 (p-value)
B
25
2+
+ 2 mM Ca
20
15
10
hsV4 ARD wt Avi
5
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
structural constituent
ribonucleoprotein of ribosome
complex RNA binding
structural molecule activity
ribosome unfolded protein binding
mRNA binding
cytoplasm rRNA binding
heterocyclic compound binding
cytoplasmic organic cyclic
ribonucleoprotein compound binding
granule single-stranded RNA binding
mRNA 5’-UTR binding
cytoplasmic
poly(A) binding
stress granule
translation regulator activity
nucleoplasm poly-pyrimidine tract binding
nucleic acid binding
supramolecular protein folding chaperone
fiber
nucleotide binding
0 5 10 15 20 25 0 5 10 15 20 25
-log10 (p-value) -log10 (p-value)
96
-log10 (p-value)
-log10 (p-value)
Chapter 4. Results
C 25
20
15
hsV4 ARD R232C Avi
10
5
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
mRNA metabolic process
ribonucleoprotein negative regulation
complex of gene expression
nuclear-transcribed mRNA
catabolic process
nucleoplasm
translational initiation
SRP-dependent cotranslational
cytoplasmic protein targeting to membrane
ribonucleoprotein intracellular protein transport
granule
cellular protein localization
peptide transport
ribosome biogenesis
organic cyclic
compound catabolic process
regulation of gene expression
peptide metabolic process
RNA processing
negative regulation of
biological process
RNA metabolic process
0 5 10 15 20 25 0 5 10 15 20 25
-log10 (p-value) -log10 (p-value)
25
D 2++ 2 mM Ca
20
15
10
hsV4 ARD R232C Avi
5
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
structural constituent
ribonucleoprotein of ribosome
complex RNA binding
mRNA binding
nucleoplasm
rRNA binding
cytoplasmic 5S rRNA binding
ribonucleoprotein mRNA 5’-UTR binding
granule ATP-dependent
helicase activity
cytoplasm nucleic acid binding
pre-mRNA binding
ubiquitin ligase inhibitor activity
heterocyclic compound binding
organic cyclic compound binding
G-rich strand
telomeric DNA binding
ATP-dependent
RNA helicase activity
single-stranded RNA binding
0 5 10 15 20 25 0 5 10 15 20 25
-log10 (p-value) -log10 (p-value)
97
-log10 (p-value) -log10 (p-value)
Chapter 4. Results
E 25
20
15
10
5 hsV4 ARD K276E Avi
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
SRP-dependent cotranslational
ribonucleoprotein
46,3 protein targeting to membrane
complex mRNA metabolic process
translational initiation
ribosome 33,5 nuclear-transcribed mRNA catabolic
process, nonsense-mediated decay
mRNA catabolic process
cytoplasm nuclear-transcribed mRNA
catabolic process
protein targeting
cytoplasmic
ribonucleoprotein translation
granule nucleobase-containing compound
catabolic process
amide biosynthetic process
nucleoplasm
peptide metabolic process
establishment of protein localization
cytoplasmic
to organelle
stress granule intracellular protein transport
protein localization to organelle
negative regulation
of gene expression
0 5 10 15 20 25 30 35 40 45 50 55 60 65
0 5 10 15 20 25
-log -log10 (p-value)10 (p-value)
F 25
2+
+ 2 mM Ca
20
15
hsV4 ARD K276E Avi
10
5
0
0 5 10 15 20
log2 fold change (biotinylated/non-biotinylated)
translational initiation
ribonucleoprotein
42,3
complex SRP-dependent cotranslational
protein targeting to membrane
mRNA metabolic process
ribosome 26,2 nuclear-transcribed mRNA catabolic
process, nonsense-mediated decay
nuclear-transcribed mRNA
cytoplasm catabolic process
translation
RNA catabolic process
cytoplasmic
ribonucleoprotein nucleobase-containing compound
catabolic process
granule
amide biosynthetic process
nucleoplasm peptide metabolic process
cellular amide metabolic process
cytoplasmic protein localization to organelle
stress granule establishment of protein localization
to organelle
cellular protein localization
intracellular protein transport
0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 45 50 55 60 65
-log10 (p-value) -log10 (p-value)
98
-log10 (p-value)
-log10 (p-value)
Chapter 4. Results
The UDMSE interactome results shown in this thesis revealed ribonucleotide binding proteins
as a new potential class of TRPV4 interacting proteins and thus indicate TRPV4 as a until now
unknown player in the regulatory mechanism of cytoplasmic ribonulceoprotein granule formation
via its cytosolic N-terminus. The following sections take a closer look on chosen TRPV4 PPIs
in vitro and in cellulo.
99
Chapter 4. Results
4.2 Birds of a feather flock together - the interaction between the
two protean proteins TRPV4 and DDX3X
As described in sub chapter 4.1, elaborate mass spectrometry (MS)-based interactome stud-
ies of hsV4N showed a significant amount of RNA-binding proteins (RBPs) as a new class of
potential TRPV4 binding partners. The composition of detected RBPs varied between different
conditions, like the presence of disease-causing mutations and/or Ca2+-supplementation. In-
triguingly, the ATP-dependent RNA-helicase DDX3X was consistently detected in all samples.
Recently, Donate-Macian et al.136 showed via a Y2H screening that DDX3X is a direct interactor
of full-length TRPV4. The MS-based interactome studies conducted in this work indicate that
DDX3X possibly binds to the TRPV4 N-terminus with the ARD (hsV4 ARD) as a minimal bind-
ing site. Log2 ratios of DDX3X in respective MS-measurements already suggested elevated
DDX3X binding-levels with mutation-bearing hsV4 ARDs (hsV4 ARD R232C and K276E, re-
spectively) in comparison to hsV4 ARD wt or hsV4N (see tables 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9
and 6.10). To conduct in vitro experiments regarding the probable direct interaction between
DDX3X and hsV4 ARD, recombinant DDX3X and TRPV4 N-terminal constructs were purified
and their structural integrity confirmed as shown in figure 4.8 (see subsections 3.7.1 and 3.7.6
for purification protocols). hsV4 ARD K276E showed heavy precipitation behavior in all sub-
sequent in vitro studies and is therefore not shown in following procedures and experiments.
Figure 4.8 (facing page): Purification of recombinant human TRPV4 N-terminal constructs and human DDX3X. A Topol-
ogy model of recombinantly expressed human TRPV4 N-terminal constructs and human DDX3X. The DDX3X construct was
optimized for future nuclear magnetic resonance spectroscopy experiments, comprising supposedly unstructured regions which
include residues aa 1 - 120 and aa 582 - 662. His6 = hexa-histidine tag (single letter code: HHHHHH), TEV = Tobacco Etch Virus
Cleavage Site (single letter code: ENLYFQG), DEAD = DEAD domain (RecA-like domain 1), HELICc = C-terminal helicase domain
(RecA-like domain 2). See figure 4.14 A for full-length DDX3X. His6-tags with preceding or subsequent TEV Cleveage Site were
removed. B SEC purifications of human TRPV4 N-terminal constructs and human DDX3X. SEC runs were performed with either
a HiLoad Superdex 200 or a HiLoad Superdex 75 pg preparative SEC column. Inlets show Coomassie stained 15 % SDS-PAGEs
of collected and concentrated fractions after SEC. x-axis: eluted volume in mL, y-axis: absorbance at 280 nm, M = marker. C
Comparison of far UV CD spectra of purified human TRPV4 N-terminal constructs and human DDX3X, respectively. Spectra were
measured at 293 K and proteins were used at 1µM concentration in either SEC Buffer with a final NaCl concentration of 30 mM
(TRPV4 N-terminal constructs) or DDX3X SEC buffer with a final concentration of 12.5 mM NaCl (DDX3X). Inlets show secondary
structure prediction performed with CAPITO.93 The predicted α-helix/β-sheet/random coil content of the purified proteins is for
hsV4N 53 %/0 %/47 %, hsV4N R232C 33 %/13 %/53 %, hsV4 ARD wt 68 %/0 %/32 %, hsV4 ARD R232C 78 %/0 %/32 % and
DDX3X 40 %/14 %/46 %. y-axis: mean residue ellipticity ([Θ]mrw,λ) in 10−3 deg cm2 dmol−1, x-axis: wavelength λ in nm.
100
Chapter 4. Results
A
1 149 398
hsV4N wt N PDB PRR ARD His
1 6 C
R232C
hsV4N R232C N PDB PRR ARD His6 C
hsV4 ARD wt N ARD His6 C
R232C
hsV4 ARD 232C N ARD His6 C
hsV4 IDR N PDB PRR TEV His6 C
121 182 404 414 544 581
DDX3X N His6 TEV DEAD HelicC C
B
MW MW
Coomassie [kDa]
200 hsV4N
350 [kDa] Coomassie
hsV4 ARD wt
75 hsV4N R232C
75 hsV4 ARD R232C
DDX3X 75
50 300
50
50
37
37
3X N 4N 37150 DX sV4 sV CD h h 232R 250
25
25
25
wt 32
C
200 R2
100
hsV4 ARD
150
100
50
50
Superdex200 Superdex75
0 0
40 50 60 70 80 90 100 40 50 60 70 80 90 100
Volume [mL] Volume [mL]
C
1 .0 random coil 1 .0 random coil
b-sheet b-sheet
60 hsV4N 0 .9 a-helix 60 DDX3X 0 .9 a-helix
hsV4N R232C 0 .8 0 .8
hsV4 ARD wt 0 .7 0 .7
hsV4 ARD R232C 0 .6 0 .6
40 40
0 .5 0 .5
0 .4 0 .4
0 .3 0 .3
20 0 .2 20 0 .2
0 .1 0 .1
0 .0 0 .0
4N 4N RDV V A AR
D X
s C t 2
C
h hs 2 w 3 DX
3
R2
3 R2 D
0 0
-20 -20
-40 -40
200 210 220 230 240 250 260 200 210 220 230 240 250 260
l [nm] l [nm]
101
3 2 -1 Abs [mAu]
q [10 deg cm dmol ] 280nm
mrw, l
sec. structure content
3 2 -1
q [10 deg cm dmol ]
mrw, l Abs [mAu]
280nm
sec. structure content
Chapter 4. Results
A B
1,2 DDX3X w/o dxRNA 35
DDX3X DDX3X w/o dxRNA
DDX3X
30 DDX3X : hsV4N 1:4
1,0
DDX3X : hsV4 ARD wt 1:4
25 DDX3X : hsV4 IDR 1:4
0,8
20
0,6
15
0,4
10
0,2 5
0,0 0
0 400 800 1200 1600 2000 2400 2800 3200 3600
C D
1,2 DDX3X 1,2 DDX3X
DDX3X : hsV4N 1:4 DDX3X : hsV4 ARD wt 1:4
DDX3X : IDR 1:4
1,0 1,0
0,8 0,8
0,6 0,6
0,4 0,4
0,2 0,2
0,0 0,0
0 400 800 1200 1600 2000 2400 2800 3200 3600 0 400 800 1200 1600 2000 2400 2800 3200 3600
time [sec] time [sec]
Figure 4.9: hsV4 ARD, but not hsV4N, decreases the DDX3X ATPase activity. A In an enzyme-coupled ATPase assay, DDX3X
showed a time-dependent duplex RNA (dxRNA)-stimulated ATPase activity (black) and a negligible ATPase activity in the absence
of dxRNA (DDX3X w/o dxRNA, dark gray), in agreement with the literature.84,142 B DDX3X ATPase activity in the presence of
several TRPV4 N-terminal constructs. Error bars indicate SDs of determined slopes after natural logarithmic conversion and linear
regression. C+D DDX3X showed decreased ATPase activity in the presence of hsV4 ARD. Interestingly, hsV4N itself and the ARD
preceding intrinsically disordered region (IDR) consisting of amino acids 1-149 of hsV4N do not demonstrate such an effect on
DDX3X. DDX3X was used at a concentration of 3µM and TRPV4 N-terminal constructs at 12µM, resulting in a 1:4 ratio. After 30 s
samples were supplemented with ATP and after 5 min with duplex RNA (dxRNA), both at a final concentration of 1µM. ATPase
activity was determined by an ATP-dependent enzyme-coupled oxidation from NADH to NAD+, resulting in an time-dependent
absorption decrease at 340 nm. Measurements were normalized to DDX3X ATPase activity. Error bars indicate SDs of three
measurements of three independent DDX3X purifications for each condition. Used TRPV4 N-terminal constructs originated from
one purification. x-axis = time in s, y-axis = extinction at 340 nm in arbitrary units. IDR was kindly provided and fully characterized
by Dr. and .143
To ensure enzymatic activity of purified DDX3X, an enzyme-coupled ATPase assay was per-
formed (see figures 4.13 C and D). In agreement with Epling et al.142, DDX3X showed a time-
dependent duplex RNA (dxRNA)-stimulated ATPase activity and a negligible ATPase activity in
the absence of dxRNA (figure 4.9 A). When supplemented with hsV4 ARD, the ATPase activity
of DDX3X significantly decreased, whereas no effect was observed upon hsV4N supplemen-
tation (see figure 4.9). Interestingly, DDX3X ATPase activity did not decrease in the presence
102
Norm. extinction [a.u.]
Norm. extinction [a.u.]
-1 -1
pmol*sec *µg
Chapter 4. Results
of the isolated TRPV4 intrinsic disordered region (hsV4 IDR), which consists of aa 1-149 of
hsV4N, preceding the hsV4 ARD. As a control, the well-characterized B. subtilis ATPase BmrA
was also supplemented with hsV4 ARD to rule out unspecific effects on the lactate dehydro-
genase and pyruvate kinase involved in this assay. No decrease in BmrA ATPase activity was
observed upon hsV4 ARD wt supplementation (see section 6.4, figure 6.3), supporting the di-
rect interaction between hsV4 ARD and DDX3X. Nevertheless the question arose, why there
is such a discrepancy between hsV4N and hsV4 ARD modulation of DDX3X ATPase activ-
ity. Cross-linking mass spectrometry (XL-MS) experiments with DSS-cross linked hsV4N and
DDX3X (see figure 4.10) revealed cross-linking between K251 of hsV4 ARD and K511 in the
C-terminal helicase domain (HelicC) of DDX3X in close proximity to motifs V (aa 501-505) and
VI (aa 525-536), which are responsible for RNA recognition (motif V) and ATP-binding (motif
VI).144
121 182 404 414 544 581
DDX3X N His TEV DEAD HelicC C6
= lysine residue
1 149 398
hsV4N wt N PDB PRR ARD His6 C
Figure 4.10: Cross-linking mass spectrometry (XL-MS) confirms a direct interaction between hsV4N and DDX3X. Green
bars indicate native lysine residues, whereas lysine residues involved in DSS-mediated cross-linking are additionally numbered.
Black lines show cross-links including hsV4N K70 and DDX3X lysine residues, red lines cross-links between hsV4N K121 and
DDX3X K491 and hsV4N K251 and DDX3X K511, respectively. XL-MS measurements were kindly carried out by M.Sc.
University of Konstanz)
XL-MS experiments of Dr. Benedikt Goretzki demonstrated an IDR-ARD interaction in the G.
gallus (chicken) TRPV4 N-terminus (ggV4N) between distinct lysine residues, indicating these
interactions not to be arbitrary but rather following certain preferred contact sites of the IDR
with the ARD.23 Amongst others, ggV4N showed an intra-domain interaction between K56IDR
and K237ARD, which could be also detected in the corresponding lysine residues K70IDR and
K251ARD in hsV4N (data not shown). It was previously shown that the TRPV4 N-termini of
chicken and human share important conserved motifs like the prolines in the PRR and show
overall similiar behavior and structural features (sharing 84.27 % amino acid identity), enabling
to transfer in vitro studies of the N-termini between the species.22 Regarding this folding behav-
ior of the IDR back to the ARD, preventing the ARD to interact with a distinct contact site with
the DDX3X HelicC domain, the difference of ATPase activity modulation between hsV4N, IDR
and hsV4 ARD could be explained. The interaction of the IDR itself seems not to play a role in
103
70
121 208
255
264
251
452
491
511
251
554
Chapter 4. Results
ATPase modulation of DDX3X. Inada et al. showed that hsV4 ARD is able to bind ATP, which
induces conformational changes in fingers 2 and 3.27
A B
DDX3X 1800000 DDX3X
1800000
l = 346 nm DDX3X:ARD 1:1
DDX3X
max DDX3X:ARD 1:4 DDX3X:ARD 1:11600000
1600000 lmax = 330 nm DDX3X:ARD 1:4
(± 1.7) l = 348 nm
lmax = 347 nm
max
1400000 (± 0.8)
1400000
1200000
1200000
lmax = 348 nm
1000000 (± 0.8)
1000000
800000
800000
l = 335 nm
600000 max
600000 (± 0.9)
400000
400000
200000 200000
lmax = 338 nm
(± 0.9)
0
0
300 320 340 360 380 400
300 320 340 360 380 400
Emission l [nm] Emission l [nm]
C 121 182 404 414 544 581
DDX3X N DEAD HelicC C
= tryptophan
Figure 4.11: hsV4 ARD interacts with duplex RNA (dxRNA)-bound DDX3X.A Tryptophan fluorescence measurements of
DDX3X and hsV4 ARD wt. B Tryptophan fluorescence measurements of DDX3X and hsV4 ARD wt in the presence of dxRNA.
Each sample was additionally supplemented with 1µM ATP, as DDX3X requires ATP for RNA binding.144 hsV4 ARD does not har-
bor tryptophan residues. DDX3X was used at a concentration of 3µM and hsV4 ARD at either 3µM (1:1 ratio) or 12µM (1:4 ratio).
Indicated samples were supplemented with duplex RNA (dxRNA) to a final concentration of 1µM. Measurements with dxRNA
were corrected due to the inner filter effect of supplemented dxRNA (see equations 4.1 and 4.2).145 Error bars indicate SDs of
three measurements of three independent DDX3X purifications for each condition. Used TRPV4 N-terminal constructs originated
from one purification. Emission maxima (λmax in nm) are listed with SDs if applicable. x-axis = emission wavelength λ in nm,
y-axis = fluorescence intensity in arbitrary units. C Topology model of recombinantly expressed and purified human DDX3X used
for tryptophan fluoresence measurements, containing four tryptophan residues (indicated by pink bars). DEAD = DEAD domain
(RecA-like domain 1), HELICc = C-terminal helicase domain (RecA-like domain 2). See figure 4.14 A for full-length DDX3X.
To exlcude unspecific DDX3X ATPase activity modulation via interactions between the dxRNA
phosphate groups and the basic surfaces of hsV4 ARD, tryptophan fluorescence measure-
ments were carried out (figure 4.11). The DDX3X construct used here harbors four tryptophan
residues (W127, W137, W421 and W570), whereas hsV4 ARD does not include any trypto-
phan residues, resulting in the DDX3X tryptophan residues to be the only reporters in these
measurements. With tryptophan fluorescence it is possible to investigate the polarity of tryp-
tophan environments in proteins, displayed by the position of the tryptophan residue emission
maxima. Polar environments result in redshifted emission maxima, hydrophobic environments
therefore in blueshifted maxima. Furthermore, the quantum yield and thus the emission inten-
sity also depends on the tryptophan’s direct environment, displaying higher emission intensities
104
Intensity [a.u.]
127
136
421
570
+ dxRNA
Chapter 4. Results
of tryptophan residues in more apolar surroundings.145 Due to inner filter effects (quenching of
tryptophan fluorescence) of the supplemented dxRNA, the obtained fluorescence spectra were
corrected according to Hellmann et al. with the following equations:145
F (x)
Correction Factor (CF) = W
FW(0)
(4.1)
The Correction Factor (CF) in equation 4.1 is obtained by the division of the fluorescence of
a tryptophane solution with 1 µM dxRNA (FW(x)) by the fluorescence of a pure tryptophane
solution (FW(0)). The tryptophan concentration here was the same as used for DDX3X in the
measurements (3 µM). The corrected tryptophan fluorescence for DDX3X (FDDX3X, corrected(x))
was then obtained by dividing the measured tryptophan fluorescence with dxRNA (FDDX3X(x))
divided by CF (see 4.2).
FDDX3X(x)FDDX3X, corrected(x) = (4.2)CF
The displayed tryptophan fluorescence measurements suggest that hsV4 ARD binds to ATP
and dxRNA-bound DDX3X. Intensity changes in figure 4.11 A are not considered significant, as
more measurements have to be carried out to receive statistical relevant results with DDX3X
and hsV4 ARD in the ratios 1:1 and 1:4, respectively. Emission spectra in figure 4.11 B hint
to a dxRNA mediated interaction between DDX3X and hsV4 ARD. Upon dxRNA addition, the
emission maximum significantly blueshifted in comparison to ATP-bound DDX3X in the absence
of dxRNA from 348 nm to 338 nm, which implies a considerable polarity change of the DDX3X
tryptophan residues towards a more hydrophobic environment. The crystal structures shown
in figure 4.12 visualize the dramatic structural change and therefore the tryptophane residues’
environmental change upon RNA binding in DDX3X.142,144 Addition of dxRNA lead to a sig-
nificant emission intensity decrease, which can be explained by the quenching effect of RNA
in tryptophane fluorescece measurements.146,147 Interestingly, upon hsV4 ARD supplementa-
tion, the DDX3X emission maxima gradually redshifted, suggesting a direct interaction between
DDX3X and hsV4 ARD. Additionally, a dramatic emission intensity increase upon hsV4 ARD
supplementation was detected, especially at a DDX3X:hsV4 ARD ratio of 1:4. These trypto-
phan residues environment changes indicate a direct hsV4 ARD interaction with DDX3X and
not unspecific binding of dxRNA by hsV4 ARD.
105
Chapter 4. Results
A
DDX3X apo DDX3X pre-unwound dsRNA
B
W421
B
W570
DEAD domain
C B
W570
N B
B W136
W421 HelicC domain
B
W136
A
A W421
W136
A
N W570
A
W421
C AW136
PDB: 6O5F
A
W570
PDB: 5E7I
B
121 182 404 414 544 581
DDX3X N DEAD HelicC C
= tryptophan
Figure 4.12: dsRNA binding changes the tryptophan environment in DDX3X dramatically. A Crystal structures of human
DDX3X in the apo state (left, PDB: 5E7I)142 and bound to double-stranded RNA (right, PDB: 6O5F )144. ATP-binding DEAD domain
is colored in light blue, the c-terminal helicase domain (HelicC) in dark blue. B Topology model of recombinantly expressed and
purified human DDX3X used for tryptophan fluoresence measurements, containing four tryptophan residues (indicated by pink
bars). DEAD = DEAD domain (RecA-like domain 1), HELICc = C-terminal helicase domain (RecA-like domain 2). See figure 4.14
A for full-length DDX3X.
XL-MS measurements were carried out without any ATP or dxRNA supplementation, as these
additions could have interfered the cross-linking reaction, showing that the DDX3X:hsV4 ARD
interaction also occurs without these components. One main advantage of XL-MS is the pos-
sibility to capture PPIs, which are very weak and/or transient.87 Therefore, the XL-MS results
combined with the rather low-resolution spectroscopic and photometric measurements indicate
that DDX3X also interacts with hsV4 ARD in the apo state in vitro, but that this interaction can
be enhanced upon ATP- and dxRNA binding. As DDX3X showed higher log2 ratios in MS-
measurements carried out in subchapter 4.1 with hsV4 ARD R232C compared to hsV4 ARD
wt or hsV4N, suggesting elevated DDX3X-binding level in the presence of this mutation, com-
plementary ATPase assays as in figure 4.9 were carried out with respective mutation bearing
TRPV4 N-terminal constructs. Interestingly, both hsV4N R232C and hsV4 ARD R232C showed
significantly higher ATPase inhibiting effects as their wild-typic complementaries (figure 4.13).
106
127
136
421
570
Chapter 4. Results
These results underline the trend that DDX3X ATPase activity inhibition by hsV4 ARD R232C
is more enhanced than by hsV4N R232C. As Dr. Benedikt Goretzki suggested23, the charge
distribution in ggV4N probably modulates the interaction between IDR and ARD. The very N-
terminal part of ggV4N is enriched with negatively charged residues, which could interact with
the overall positively charged residues of the ARD finger 1 and 2. Arg232 in hsV4 ARD resides
in finger 2 and furthermore builds a salt bridge to Glu183 in finger 1, which was observed in
the crystal structure of the isolated hsV4 ARD.27 The neutral mutation R232C could disrupt the
overall basic patch in hsV4 ARD which might be required for the intra-domain IDR-ARD inter-
action. Additionally, the disruption of the R232-E183 salt bridge could influence the dynamics
of finger 1 and 2 of hsV4 ARD, leading to altered conformational hierarchies in the IDR-ARD
interaction. Thus, the R232C mutation could lead to a decreased IDR-mediated masking of the
probable DDX3X interaction site of hsV4 ARD.
A B C
DDX3X alone DDX3X alone DDX3X
DDX3X : hsV4N 1:4 DDX3X : hsV4 ARD wt 1:4 DDX3X : hsV4N 1:4
DDX3X : hsV4N R232C 1:4 DDX3X : hsV4 ARD R232C 1:4 DDX3X : hsV4 ARD wt 1:4
DDX3X : IDR 1:4 DDX3X : IDR 1:4
DDX3X : hsV4N R232C 1:4
1,2 1,2 35
DDX3X : hsV4 ARD R232C 1:4
30
1,0 1,0
25
0,8 0,8
20
0,6 0,6
15
0,4 0,4
10
0,2 0,2
5
0,0 0,0 0
0 400 800 1200 1600 2000 2400 2800 3200 3600 0 400 800 1200 1600 2000 2400 2800 3200 3600
time [sec] time [sec]
Figure 4.13: DDX3X ATPase activity inhibition is enhanced in the presence of neuropathy-causing TRPV4 R232C mutation.
A + B An enzyme-coupled ATPase assay reveals an inhibitory effect of both, hsV4N R232C and hsV4 ARD R232C, onto the
ATPase activity of DDX3X.84 DDX3X was used at a concentration of 3µM and TRPV4 N-terminal constructs at 12µM, resulting into
a 1:4 ratio. After 30 s samples were supplemented with ATP and after 5 min with duplex RNA (dxRNA), both at a final concentration
of 1µM. ATPase activity was determined by an ATP-dependent enzyme-coupled oxidation from NADH to NAD+, resulting in
an time-dependent absorption decrease at 340 nm. Measurements were normalized to DDX3X ATPase activity (DDX3X). Error
bars indicate SDs of three measurements of three independent DDX3X purifications for each condition. Used TRPV4 N-terminal
constructs originated from one purification. The TRPV4 intrinsic disordered region (IDR), consisting of aa 1-149 preceding the
ARD of hsV4N, was kindly provided and fully characterized by Dr. i23 and M. Sc. .143 x-axis
= time in s, y-axis = extinction at 340 nm in arbitrary units C DDX3X ATPase activity in the presence of several TRPV4 N-terminal
constructs. Error bars indicate SDs of determined slopes after natural logarithmic conversion and linear regression.
Additionally to the Y2H screen showing direct interaction between the full-length TRPV4 chan-
nel and DDX3X, Donate-Macian et al. performed co-IPs with full-length TRPV4 and DDX3X
co-transfected in HEK293 cells. It was shown that DDX3X immunoprecipitates with TRPV4 and
vice versa. To evaluate these in cellulo experiments, co-IPs were performed with HEK293T
107
Norm. extinction [a.u.]
-1 -1
pmol*sec *µg
Chapter 4. Results
cells co-transfected with full length human TRPV4 with a c-terminal GFP-tag (hsTRPV4-cGFP)
and full-length human DDX3X with a n-terminal V5-tag (hsDDX3X-nV5, see figure 4.14 A for a
full-length DDX3X topology model of the here used construct compared to the recombinantly
expressed and purified DDX3X used for the here shown in vitro experiments). Furthermore, to
investigate the effects of TRPV4 disease-causing mutations on DDX3X interaction in cellulo,
co-transfection and -IPs with hsTRPV4 R232C-cGFP and hsDDX3X-nV5 were also performed.
Transfections with hsTRPV4 K276E-cGFP resulted in severe cell death (data not shown). Fig-
ure 4.14 B and C show two western blots after co-IPs. Both western blots showed co-IP
of hsDDX3X-nV5 with hsTRPV4 R232C-cGFP. With hsTRPV4-cGFP, only a weak band for
hsDDX3X-nV5 in figure 4.14 B was detected.
A
149 398 871
hsTRPV4-cGFP N PDB PRR ARD GFP C
R232C
hsTRPV4 R232C-cGFP N PDB PRR ARD GFP C
1 12 22 38 44 132 182 404 414 544 607 662
hsDDX3X-nV5 N V5 NTE DEAD HELICc CTE C
Recombinantly expressed and purified DDX3X
B C D
1,2
250 250
150 150 TRPV4
100 100
75 75 1,0
Heavy chain
50 50
37
37 0,8
Light chain
250 250 0,6
150 150
100 100
75 75
Heavy chain 0,4
50 50
37
37
Light chain 0,2
DDX3X 0,0
75 75
50 Heavy chain50
Input (5 %) Co-IP (a-GFP) Input (5 %) Co-IP (a-GFP)
Figure 4.14: Co-Immunoprecipitations (co-IPs) of full-length human TRPV4-cGFP and TRPV4 R232C-cGFP, respectively,
in HEK293T cells show an increased DDX3X-V5 pulldown with the neuropathy-causing TRPV4-R232C mutation. A Topol-
ogy models of human full-length TRPV4 constructs and DDX3X used for co-expression in HEK293T cells. For TRPV4, important
N-terminal domains, namely the proline rich region (PRR), phosphoinositide-binding domain (PBD) and ankyrin repeat domain
(ARD), are highlighted. GFP = Green-fluorescent protein, V5= V5-tag (single letter code: GKPIPNPLLGLDST), NES = nuclear
export sequence, elF4E = binding site of transcription factor elF4E, NTE= N-terminal extension, DEAD = DEAD domain (RecA-like
domain 1), HELICc = C-terminal helicase domain (RecA-like domain 2), CTE = C-terminal extension.148 B + C Two α-GFP co-IPs
with full-length human TRPV4-cGFP and TRPV4 R232C-cGFP, respectively, co-transfected with hsDDX3X-nV5 in HEK293T cells
show an increased hsDDX3X-nV5 pulldown with the neuropathy-causing TRPV4-R232C mutation. To rule out unspecific interac-
tions, soluble GFP was co-transfected with respective TRPV4 construct. D Densitometric quantification of co-immunoprecipiated
hsDDX3X-nV5, normalized to immunoprecipitated hsTRPV4-cGFP of respective condition. Error bars indicate SD. Densitometry
was performed with Fiji.
108
hsDD
G XF 3P X-nV
h 5s
G DF DP X3X-
h ns VT 5R +P
h Vs 4D -hs D
cG
T X FR 3P X
P
-
h Vs 4
nV
T -R c
5
P G
+
V Fh 4 Ps
h DD Rs 2T 3R X 2P 3X C
M V -n
-cG
[ 4 Vk 5 FD R + Pa 2] 32C-c
h GsD FD P
G XF 3P X-nV
h 5s
G DF DP X3X-
h ns VT 5R +P
h V4 NESsD -hs D
cG
T X FR 3P X
P
V -h 4 ns VT - 5R cG +P F elF4E
h V Ps 4
h DsT D
R
X 23RP 3
2
X C
V -n -c4 V GR F2 5 + P32C-cGFP
hsDDX
G 3F XP -nV
h 5s
G DF DP X3X-
h ns VT 5R +P
h Vs 4-c
h DD GsT X FR 3 PP X
h V
-
4 nsT -
V
c 5R +P GF
h V4 Ps
h D Rs 2T D 3R XP 3
2
M X
C
V -n -c
[k 4 R V
G
D 2 5
F
+ Pa] 32C-c
h GsD FD PX
G 3F XP -nV
h 5s
G DF DP X3X-
h ns VT 5R +P
h Vs 4-c
h D Gs DT X FR 3X PPV -h ns 4- VTR c
5
P G
+
V Fh Ps 4
h D Rs 2T DR X
32
P 3X CV -n -c4 GR V F2 5 P3 +2C-cGFP
a-V5, 5 min a-GFP, 5 min a-GFP, 30 sec
DDX3X
IP band intensity
hsTRPV4-nGFP
hs
h Ds DT XR 3P XV -4 n- Vc 5G +FP
h
R s2 T3 R2 PC V-c 4GFP
hsDD
h X
R s
3X
2 TR -3 n2 P VC V 5- +c 4GFP
Chapter 4. Results
Even though the amount of co-immunoprecipitated hsDDX3X-nV5 is low, the in the ATPase as-
says (figure 4.13) observed trend of a stronger DDX3X-TRPV4 R232C interaction compared
to wild-type TRPV4 persisted. In the last few years, pathological stress granule formation
more and more emerged to be one possible cause of neurodegenerative diseases such as the
TRPV4-associated Charcot-Marie-Tooth Type 2C disease. These membraneless organelles
form in cellulo upon oxidative and hyperosmotic stress, amongst others, as a consequence of
stress-induced translational shutdown and polysome disassembly.121,149 Stress granules are
composed mainly from RNA-binding proteins like DDX3X and form highly dynamic structures
upon acute stress, whereas chronic cellular stress is proposed to induce pathologically stable
complexes that might promote the formation of aggregates.149–151
Based on the elevated DDX3X-TRPV4 R232C interaction shown in subchapter 4.1.2 and in fig-
ures 4.13 and 4.14, fluorescence microscopy experiments with HEK293T cells co-transfected
with the same constructs used as in the co-IPs in figure 4.14 were performed to investigate prob-
able foci formations of DDX3X in the presence of TRPV4 and/or neuropathy causing TRPV4
R232C (figure 4.15). Transiently transfected HEK293T cells showed expected subcellular lo-
calization of respective proteins, namely hsDDX3X-nV5 in the cytosol and hsTRPV4-cGFP in
the plasma membrane. In the case of hsTRPV4 R232C-cGFP, beside the plasma membrane
localization also GFP-containing foci were observed. For hsDDX3X-nV5 alone an uniform
cytosolic distribution was observed, whereas in the presence of hsTRPV4-cGFP densifica-
tions formed. These hsDDX3X-nV5 densifications were more pronounced and spatially dis-
tinct as foci in the presence of hsTRPV4 R232C-cGFP. hsDDX3X-nV5 and hsTRPV4-cGFP or
hsTRPV4 R232C-cGFP co-expression did not alter the subcellular localization of both TRPV4
constructs. These results indicate an influence of TRPV4 on DDX3X-containing foci forma-
tion in cellulo. Valentin-Vega et al. showed that medulloblastoma-associated mutations in
DDX3X, leading to deficiency in RNA-stimulated ATP-hydrolysis of DDX3X, spur stress gran-
ule hyperassembly.119,142 Therefore, not only the increased basal Ca2+ influx levels due to
the neuropathy causing R232C mutation could lead to abnormal stress granule formations, but
also the elevated ATPase inhibition due to an increased DDX3X interaction with the mutated
hsV4 ARD. Donate-Macian et al. showed a decreased DDX3X co-IP with TRPV4 in Huh7 cells
Figure 4.15 (facing page): Fluorescence microscopy images of HEK293T cells co-transfected with TRPV4-GFP (green) and
DDX3X-V5 (magenta) reveal cytosolic DDX3X densifications (white arrow heads), especially in the presence of TRPV4-
R232C-GFP. V5-tag of DDX3X was detected via mouse anti-V5 primary antibody and subsequent Alexa Fluor™647-coupled
anti-mouse secondary antibody staining. Nuclei were stained with DAPI. Scale bar = 10µm. Images were taken with a Zeiss Axio
Observer Z1 inverted widefield microscope.
109
Chapter 4. Results
DDX3X-V5 hsTRPV4-cGFP DAPI
110
hsDDX3X-nV5 hsTRPV4-cGFP hsDDX3X-nV5 + hsTRPV4 R232C-cGFP hsDDX3X-nV5 +
hsTRPV4-cGFP hsTRPV4 R232C-cGFP
Chapter 4. Results
upon treatment with the TRPV4 activator GSK1016790A (GSK-101), inducing TRPV4-mediated
Ca2+-influx. Additionally, TRPV4-mediated Ca2+-influx, activated either via hypotonicity or the
TRPV4 agonist GSK-101, induced partial shuttling of DDX3X into the nucleus in HEK293 cells
via a Ca2+/Calmodulin-dependent pathway. This shuttling behavior was not observed with the
gain-of-function R232C mutation in TRPV4 in this thesis (figure 4.15).54,136 In summary, the
results by Donate-Macian et al. indicate a disruption of the TRPV4-DDX3X PPI upon TRPV4
activation.136 In combination with the results shown in this thesis, the increased TRPV4 R232C-
DDX3X PPI seems to be Ca2+-influx independent, as TRPV4 R232C was previously shown to
exhibit elevated basal Ca2+-influx activity.54 The TRPV4 R232C-DDX3X PPI seems to be ele-
vated due to mutation-induced electrostatic and structural changes within and between the two
proteins. These findings underline the importance of stress granule homeostasis and the severe
consequences of dysregulation in stress granule dynamics, which ultimately leads to neuronal
cell death.119 The data shown in this thesis reveal TRPV4 as a potential communication hub for
downstream processes involved in stress granule formation.
111
Chapter 4. Results
4.3 It is all about humanity - an in cellulo study of the interaction
between human TRPV4 and PACSIN1-3
Besides the six ankyrin repeats organized in an ankyrin repeat domain (ARD), the hsV4N con-
tains a proline rich region (PRR, aa 132 – 144) which is necessary for the interaction with
PACSIN3 (Protein kinase C and casein kinase substrate in neurons 3), one of the few known
direct interactors of TRPV4.70 All three PACSIN isoforms consist of a F-BAR domain, a linker
domain and a SH3 domain, of which the latter interacts with the TRPV4 PRR (see figure 4.16
F).22,70,71 Previous experiments evaluated the physiological role of PACSIN3 in TRPV4 modu-
lation, where it was shown that the relative plasma membrane amount of TRPV4 is enhanced
by PACSIN3, but not by PACSIN1 and 2, despite of co-IPs of all three mouse PACSINs with rat
TRPV4.70 Furthermore, D’hoedt et al. showed that PACSIN3 desensitizes TRPV4 and inhibits
its activation by cell swelling induced via a hypoosmotic solution and heat, but not by chemi-
cal stimuli like the known TRPV4 activator 4α-phorbol 12,13-didecanoate (4α-PDD). Although
these previous experiments comprehensively evaluated the physiological role of PACSIN3 in
TRPV4 modulation, data about the roles of PACSIN1 and PACSIN2 are lacking.70,71 Also, be-
fore mentioned experiments where performed with rodent proteins (mouse PACSINs and rat
TRPV4 or mouse TRPV4) in a human expression system (HEK293T cells). To elucidate the
role of all PACSINs in TRPV4 modulation, in this work human full-length PACSINs and TRPV4
were co-expressed in HEK293T cells to gain a comprehensive and species consistent view on
the physiological role of these PPIs. Following the initial studies of D’hoedt et al.71 showing
dampened TRPV4-mediated Ca2+-influx upon hypotonicity by PACSIN3 in HEK293T cells, a
Fura-2 AM-based Ca2+-influx assay was performed (see figure 4.16 A). HEK293T cells co-
transfected with human full-length TRPV4 and PACSIN1 or PACSIN3, respectively, show signif-
icant dampened TRPV4-mediated Ca2+-influx in comparison to cells transfected with TRPV4
alone or co-transfected with PACSIN2. Furthermore, PACSIN1 and PACSIN3 dampen the basal
Ca2+-influx activity of TRPV4 (t=0). Figure 4.16 B shows that PACSIN1 and 3, but not PAC-
SIN2, co-immunoprecipitate with TRPV4, which is in disagreement with the co-IPs performed
with the rodent PACSIN orthologues and TRPV4.70 As expression levels of all three PACSINs
in figure 4.16 are on a comparable level, these results underline the difference of PPIs between
protein orthologues across species, even in the context of high protein identities (see section
6.5).
112
Chapter 4. Results
A B
2,4 2,4
Hypotonicity GSK-101
2,2 2,2
2,0 2,0
1,8 1,8
1,6 1,6
1,4 1,4
1,2 1,2
1,0 1,0
0,8 0,8
0,6 0,6
0,4 0,4
0,2 EV TRPV4-FLAG 0,2PAC1-V5 PAC2-V5 PAC3-V5 EV TRPV4-FLAG PAC1-V5 PAC2-V5 PAC3-V5
+TRPV4-FLAG +TRPV4-FLAG
0,0 0,0
0 100 200 300 400 0 100 200 300 400
time [s] time [s]
*
C 2,0D
1,8
1,6
M 1,4
[kDa]
1,2
150
1,0
100
0,8
75
0,6
0,4
50
0,2
37
0,0
Input (5 %) co-IP (a-FLAG)
E F
PACSIN1-V5 PACSIN2-V5 PACSIN3-V5
1 444
PACSIN1 N V5 F-BAR SH3 C
1 486
PACSIN2 N V5 F-BAR SH3 C
1 424
PACSIN3 N V5 F-BAR SH3 C
1 871
TRPV4
N FLAG PDB PRR ARD C
-FLAG
TRPV4-FLAG/PACSIN-V5/DAPI
Figure 4.16: Human PACSIN1 and 3, but not PACSIN2, dampen human TRPV4-mediated Ca2+-influx in transiently co-
transfected HEK293T cells upon hypotonicity. A HEK293T cells transiently co-transfected with TRPV4-FLAG and PACSIN1-
3-V5, respectively, were loaded with the calcium indicator Fura-2 AM and treated with hypotonic saline (30 mM NaCl) at time = 0.
Intracellular calcium levels were measured by acquiring 340 nm/380 nm ratio images every 10 s for 480 s. EV = transfection with
empty vector. Averages represent n = 3, where each measurement was conducted with 4 coverslips with 20-30 cells per coverslip
per condition. Error bars indicate SD. x-axis = time in s, y-axis = Fura 2-AM 340 nm/380 nm wavelength ratio. B HEK293T
cells transiently co-transfected with TRPV4-FLAG and PACSIN1-3-V5, respectively, were loaded with the calcium indicator Fura-
2 AM and treated with 30 nM of the TRPV4 agonist GSK-101 at t = 0. Fore more experimental details see subfigure A. C
Representative α-FLAG co-immunoprecipitation of TRPV4-FLAG and PACSIN1-3-V5, respectively, co-transfected in HEK293T
cells show co-immunoprecipitation of PACSIN1 and 3, but not PACSIN2, with TRPV4-FLAG.D Densitometric quantification of co-
immunoprecipiated PACSINs, normalized to immunoprecipitated TRPV4-FLAG of respective condition. Paired two-tailed t-test, n=3
from three independent co-IPs, *p ≤ 0.05. Error bars indicate SD. Densitometry was performed with Fiji. E Immunofluorescence
images of HEK293T cells co-transfected with TRPV4-FLAG (red) and PACSIN1-3-V5 (green), respectively, show proper cellular
distribution of overexpressed constructs with TRPV4-FLAG in the plasma membrane and PACSIN1-3-V5 in the cytosol. FLAG-
tag was detected with rabbit α-FLAG and subsequent α-rabbit Alexa Fluor 555 staining. V5-tag was detected with mouse α-V5
and subsequent α-mouse Alexa Fluor 488 staining. Nuclei were stained with DAPI. Scale bar = 10µm. Images were taken
with a Zeiss LSM800 Confocal Microscope. F Topology model of expressed PACSIN constructs. V5= V5-tag (single letter code:
GKPIPNPLLGLDST), FLAG = FLAG tag (single letter code: DYKDDDDK)
113
Fura-2 ratio (340/380)
TR
P PA V
C 4
1 -- F
P V
LA
A 5 G
C  
1 +-
T V
P R
5
A P
C V
2 4- -V F5 LP AA G
C  
3 +-V
T 5
P RA P
C V
3 4- -V F5 LAG
 +
TR
PV4-
T FR L
P APA V G
C 4
1 -- F
P V
L
A 5 A
C G
1  +-V
T 5
P RA P
C V
2 4-F
P -V LA 5 A
C G
3  - +V
T 5
P RA P
C V
3 4- -V F5 LAG
 +
a-V5, 30 sec a-FLAG, 30 sec
Fura-2 ratio (340/380)
TR
P PA V
C 4
1 -- FV L5 AG
 +
T
P RA P
C V
2 4- -V F5 LAG
 +
T
P RA P
C V
3 4- -V F5 LAG
 +
Chapter 4. Results
To confirm functional expression of TRPV4, complementary Ca2+-influx experiments were per-
formed with the potent TRPV4 agonist GSK-101, showing no PACSIN-mediated TRPV4 Ca2+-
influx dampening (see figure 4.16 C).70,152,153 Additionally, immunofluorescence images in fig-
ure 4.16 D show proper subcellular distributions of expressed constructs in cellulo.
PACSIN3 mRNA transcripts were mainly found in skeletal and heart muscle tissue, whereas
PACSIN2 transcripts were detected ubiquitously and PACSIN1 transcripts showed neurospeci-
ficity.154 Interestingly, co-expression of the neuropathy-causing TRPV4 R269C mutation and
PACSIN1 showed a strong trend towards a loss of TRPV4-mediated Ca2+-influx inhibition
in HEK293T cells upon hypotonicity, whereas PACSIN3 still retained its desensitizing role in
TRPV4 regulation (figure 4.17). In agreement with Woolums et al., Ca2+-influx experiments
indicate higher basal TRPV4 R269C Ca2+-influx activity compared to wild-typic TRPV4 (t = 0)
.53 Despite the loss of the PACSIN1-desensitizing function upon stimuli induced TRPV4 R269C
activation, co-expression of PACSIN1 with TRPV4 R269C still seems to dampen basal TRPV4
mediated Ca2+-influx (t=0). To substantiate these trends, additional Ca2+-influx assays are
required. A first co-IP hints towards a loss of PACSIN1-binding to TRPV4 R269C (figure 4.17
B). These results indicate a neuron-specific consequence of the neuropathy-causing TRPV4
R269C mutation, also regarding the above mentioned tissue specific mRNA transcription of the
three PACSIN isoforms. The mechanisms of PACSIN-mediated TRPV4 modulation still remain
elusive until this date. Neurons are more vulnerable towards chronic Ca2+-influx compared to
other cell types due to their unique cellular structure, which lead to higher sensitivity towards
Ca2+-dyshomeostasis with excitotoxicity as a possible consequence .155 As the desensitizing
function of PACSIN1 precedes upon the TRPV4 R269C mutation in neurons, other tissues are
possibly still able to regulate TRPV4-mediated Ca2+-influx via PACSIN3 and/or other until now
not known TRPV4 regulating mechanisms.
To shed light on the PPI between the PACSINs and the TRPV4 PRR, in chemical shift pertur-
bation assays using nuclear magnetic resonance (NMR) spectroscopy, Goretzki et al. probed
structural changes of the different PACSIN SH3 domains upon interacting with the TRPV4-
PRR.22 All three SH3 domains interact with the TRPV4-PRR in a highly comparable manner.
These experiments showed that the SH3 domains of PACSIN1, 2 and 3 interact with the TRPV4-
PRR through the same binding interfaces and have highly similar binding affinities. However,
co-IPs (figure 4.16 B) showed that PACSIN3 binds better to TRPV4 than PACSIN1, and that
PACSIN2 does not bind to TRPV4 in cellulo at all. This strongly hints towards a difference
in the TRPV4-PACSIN3 interaction compared to its interaction with PACSIN1 in the context of
full-length PACSINs and full-length TRPV4.
114
Chapter 4. Results
A B
1,6
Hypotonicity
M
[kDa]
1,4
150
100
1,2
75
1,0 50
37
0,8
0,6
150
100
0,4
75
0,2 EV TRPV4-FLAG PAC1-V5 PAC2-V5 PAC3-V5 50
TRPV4 R269C-FLAG +TRPV4 R269C-FLAG
37
0,0
0 100 200 300 400
time [s]
Figure 4.17: Human PACSIN1 does not dampen TRPV4-mediated Ca2+-influx in HEK293T cells transiently transfected with
TRPV4 R269C upon hypotonicity. A HEK293T cells transiently co-transfected with TRPV4-FLAG or neuropathy-causing TRPV4
R269C-FLAG and PACSIN1-3-V5, respectively, were loaded with the calcium indicator Fura-2 AM and treated with hypotonic
saline (30 mM NaCl) at time = 0. Intracellular calcium levels were measured by acquiring 340 nm/380 nm ratio images every
10 s for 480 s. EV = transfection with empty vector. Averages represent n = 2, where each measurement was conducted with 4
coverslips with 20-30 cells per coverslip per condition. x-axis = time in s, y-axis = Fura 2-AM 340 nm/380 nm wavelength ratio. B
α-FLAG co-immunoprecipitation of neuropathy-causing TRPV4 R269C-FLAG and PACSIN1-3-V5, respectively, co-transfected in
HEK293T cells show only co-immunoprecipitation of PACSIN3 with TRPV4, but not PACSIN1 and 2.
Since Goretzki et al. have excluded the SH3 domain as the origin of these differences, the
next step was to focus on the F-BAR domains.For this, the three F-BAR domains of PACSIN1-3
where exchanged while retaining the linker and SH3 domain of the original PACSIN via Gib-
son Assembly.79,80 Of these originally six cloned constructs, only three showed expression in
HEK293T cells (see figure 4.18 C for detailed chimera construct composition). Figure 4.18 A
shows the results of Fura-2 AM-based Ca2+-influx assays performed with HEK293T cells co-
transfected with TRPV4 and respective PACSIN chimeras treated with hypotonic saline. Both
PACSIN chimeras that combine the PACSIN1 linker and SH3 domain with either the PACSIN2
or PACSIN3 F-BAR domain (PACSIN2+1 and PACSIN3+1, figure 4.18 C), respectively, show
no regulation of TRPV4-mediated Ca2+-influx upon hypotonicity. Strikingly, PACSIN chimera
PACSIN2+3, consisting of the PACSIN2 F-BAR domain with PACSIN3 linker and SH3 domain,
shows comparable Ca2+-influx dampening to PACSIN3-mediated dampening (figure 4.16 A).
Co-IPs with TRPV4-FLAG and N-terminally V5-tagged PACSIN chimeras could not show a
pulldown of any PACSIN chimera with TRPV4. Immunofluorescence images (figure 4.18 D)
confirmed proper cellular distribution of the PACSIN chimeras in the cytosol and TRPV4 in the
plasma membrane.
115
Fura-2 ratio (340/380)
Input (5 %) Co-IP (a-FLAG)
TR
PV4 
P RA 2
C 6
1 9- CV -
PA 5
FLA
C G
2-V
P 5A
C
3-V5
T
P RA P
C V
1 4
T -
 
V R2
P R
5 6
A P
9
C V
C
-F
2 4
T -
 
V R
L
2 AR 5 6 GP  A P 9V C +C 4 -3- R
FL
a-V5, a-FLAG, a-V5, a-FLAG, V5 26
A
9 G 
30 sec 30 sec +30 sec 30 sec C-FLA
G
 +
Chapter 4. Results
A B
Hypotonicity
1,6
1,4
M
1,2 [kDa]
150
1,0 100
75
0,8
50
0,6
37
0,4
Input (5 %) co-IP (a-FLAG)
0,2 EV TRPV4-FLAG PAC2+1-V5 PAC3+1-V5 PAC2+3-V5
+TRPV4-FLAG
0,0
0 100 200 300 400
time [s]
C D
1 11 282 385 444 PACSIN2+1-V5 PACSIN3+1-V5 PACSIN2+3-V5
PACSIN2+1 N V5 F-BAR Linker SH3 C
PACSIN2 PACSIN1
1 10 280 385 444
PACSIN3+1 N V5 F-BAR Linker SH3 C
PACSIN3 PACSIN1
111 282 363 424
PACSIN2+3 N V5 F-BAR Linker SH3 C
PACSIN2 PACSIN3
TRPV4-FLAG/PACSIN-V5/DAPI
Figure 4.18: PACSIN chimeras hint towards different regulation modes between PACSIN1 and PACSIN3 on TRPV4-
mediated Ca2+-influx in HEK293T cells. A HEK293T cells transiently co-transfected with TRPV4-FLAG and respective PACSIN
chimera shown in subfigure C, respectively, were loaded with the calcium indicator Fura-2 AM and treated with hypotonic saline (30
mM NaCl) at time = 0. Intracellular calcium levels were measured by acquiring 340 nm/380 nm ratio images every 10 s for 480 s.
EV = transfection with empty vector. Averages represent n = 3, where each measurement was conducted with 4 coverslips with
20-30 cells per coverslip per condition. Error bars indicate SD. x-axis = time in s, y-axis = Fura 2-AM 340 nm/380 nm wavelength
ratio. B Representative α-FLAG co-immunoprecipitation of TRPV4-FLAG and respective PACSIN chimera (see subfigure C) co-
transfected in HEK293T cells show no co-immunoprecipitation of the PACSIN chimeras with TRPV4-FLAG. C Topology model of
expressed PACSIN chimeras. For this the three F-BAR domains of PACSIN1-3 where exchanged while retaining the linker and SH3
domain of the original PACSIN. Of these originally six cloned constructs, only the pictured three showed expression in HEK293T
cells. V5= V5-tag (single letter code: GKPIPNPLLGLDST), FLAG = FLAG tag (single letter code: DYKDDDDK). D Immunofluo-
rescence images of HEK293T cells co-transfected with TRPV4-FLAG (red) and respective PACSIN chimera show proper cellular
distribution of overexpressed constructs with TRPV4-FLAG in the plasma membrane and the PACSIN chimeras in the cytosol.
FLAG-tag was detected with rabbit α-FLAG and subsequent α-rabbit Alexa Fluor 555 staining. V5-tag was detected with mouse
α-V5 and subsequent α-rabbit Alexa Fluor 488 staining. Nuclei were stained with DAPI. Scale bar = 10µm. Images were taken
with a Zeiss LSM800 Confocal Microscope.
These results indicate different TRPV4-regulation modes between PACSIN1 and PACSIN3.
Whereas PACSIN1 seems to need its F-BAR domain, PACSIN3 seems to regulate TRPV4-
mediated Ca2+-influx either F-BAR-independently or the PACSIN2 F-BAR domain is sufficient
enough to substitute the PACSIN3 F-BAR domain (see also section 6.5 for mutliple sequence
alignment between human PACSIN F-BAR domains, indicating a higher sequence identity be-
tween the PACSIN2 F-BAR domain and PACSIN3 F-BAR domain than between the PACSIN1
F-BAR domain and PACSIN3 F-BAR domain). Cuajungco et al. showed that PACSIN3 lacking
116
Fura-2 ratio (340/380)
TR
PV4
T -R FP LA P A
C V G
2 4-
T + FR 1 LP - AA P V
C V
5 G
4  +3+ -F
T 1 LR -V AP P 5 GA V  +C
2 4+ -F3 L-V A5 G +
TR
PV4
T -R FP LA P A
C V G
2 4+ -T F
P R
1 L
P -V AA 5 G
C V4  +3+ -F
T 1 LR -V APA P 5
G
C V
 +
2 4+ -F3 L-V A5 G +
a-V5, 30 sec a-FLAG, 30 sec
Chapter 4. Results
the F-BAR domain lost its ability to dampen TRPV4-mediated Ca2+-influx upon hypotonicity in
HEK293T cells.70 This would hint towards the ability of the PACSIN2 F-BAR domain to substi-
tute the PACSIN3 F-BAR domain. BAR domain-containing proteins like PACSIN1-3 are known
to induce various forms of membrane deformations.156,157 Membrane deformation experiments
conducted by Goh et al. showed that the isolated F-BAR domains of PACSIN2 and PACSIN3
produced similar membrane morphologies after incubation with liposomes. Whereas PAC-
SIN2 and 3 F-BAR proteins mainly induced formation of vesicular structures and wide tubules,
the PACSIN1 F-BAR domain additionally induced narrow tubule formation. Additionally, Bai
et al. showed in liposome tubulation assays that PACSIN3 induced low curvature tubulation
with mainly tubule formation diameters from 90 nm to 110 nm, whereas PACSIN1 also addi-
tionally formed high curvature tubules, ranging from 10 nm to 200 nm. In this case, PACSIN2
again showed an intermediate behavior between PACSIN1 and 3, where PACSIN2-induced
tubules showed a diameter range like PACSIN1, but the relative number of low-curvature tubes
is higher compared to PACSIN1. It was shown previously that TRPV4-activation indeed is de-
pendent from the surrounding membrane fluidity and stiffness and thus different degrees of
membrane curvatures could also play a regulatory role in TRPV4 regulation.158 As the different
PACSIN isoforms serve different levels and forms of membrane deformations, PACSIN isoform
dependent TRPV4 regulation via the F-BAR mediated membrane microenvironment seems to
be an explanation for the possible different TRPV4-regulation modes between PACSIN1 and
3.156,157,159,160 These findings also underline the probable ability of the PACSIN2 F-BAR do-
main to induce similar membrane curvatures like the PACSIN3 F-BAR domain. Therefore the
PACSIN2 F-BAR domain may probably serve comparable local membrane environments for
TRPV4 in the context of the PACSIN chimera 2+3.156,160 Membrane deformation activity as-
says with all three murine full-length PACSIN isoforms showed reduced membrane deformation
activity compared to the isolated F-BAR domains, whereby full-length PACSIN1 showed a sig-
nificantly higher activity loss than PACSIN3. PACSIN2 showed again an intermediate behavior,
where an activity decrease was also observed, but this decrease was not as pronounced as
for PACSIN1. Membrane deformation activity reduction might occur due to the autoinhibition by
the SH3 domain binding on the F-BAR domain, preventing the F-BAR domain to interact with
membranes. This particular autoinhibition was shown for PACSIN1 in a X-ray crystal structure
of murine PACSIN1 (PDB: 2X3X), where a negatively charged triple motif (Q396, E397, E400)
in the SH3 domain interacts with a basic lysine patch (K141, K145, K148) in the F-BAR do-
main. This was confirmed with site-directed mutagenesis with subsequent pull-down assays
in cellulo.156,157 As the substrate-binding site of the SH3 domain overlaps with the SH3/F-BAR
117
Chapter 4. Results
domain interacting site, it was shown that the dynamin-PRR interrupts PACSIN1 autoinhibition
and induces PACSIN1-mediated lipid tubulation.156 This autoinhibition mechanism was also
proposed for PACSIN2 and 3, as the SH3 and F-BAR domains amongst the isoforms are highly
conserved, including the SH3/F-BAR domain interaction sites. Indeed, Goretzki et al. could
show via NMR spectroscopy that the PACSIN3 SH3 domain interacts with the PACSIN3 F-BAR
domain and that a QEE/RRR mutation in the PACSIN3 SH3 domain abrogated this interaction.
Furthermore, the interaction between PACSIN3 SH3 and F-BAR seemed to be comparably
weak and could be released with a TRPV4-PRR containing peptide in a similar manner to
the dynamin-PRR-mediated autoinhibition-release of PACSIN1.23,156 The strength between the
SH3 and F-BAR domain interaction of PACSIN1 and 2 still have to be determined and variations
in respective KD values could serve as another explanation for the different TRPV4-mediated
behaviors between the three PACSIN isoforoms and the possible ability of the PACSIN2 F-BAR
domain to substitute the PACSIN3 F-BAR domain in the chimera PACSIN2+3. Expression of
the PACSIN chimera consisting of PACSIN1 F-BAR domain and PACSIN3 linker and SH3 do-
main was not possible. Therefore it remains elusive, if the PACSIN1 F-BAR domain could also
substitute the PACSIN3 F-BAR domain like occured in PACSIN chimera 2+3.
Another explanation for the different TRPV4-regulation outcomes of the PACSIN chimeras could
be different protein interactomes of PACSIN1 and 3, due to their different tissue distributions and
minor differences in amino acid sequences.154 Goretzki et al. proposed that PACSIN3 possi-
bly binds to TRPV4 as a dimer, where the F-BAR domains mediate the PACSIN3 dimerization.
One SH3 domain therefore would interact with the TRPV4 PRR, whereas the other SH3 domain
could recruit additional TRPV4 binding partners.
Furthermore, PACSIN3 is the only PACSIN isoform that contains a proline rich region in its linker
region (aa 337-345), with which other proteins could interact with. PACSIN chimera 3+2 thus
seems still to be able to bind to the TRPV4 PRR via its SH3 domain, exposing the PACSIN3
PRRs in the linker domains and the other SH3 domain for further protein interactions to form
a multicomponent TRPV4 signaling complex. This could be an additional determinant for a
PACSIN3- and therefore tissue specific protein interactome, which leads to a robust PACSIN3-
TRPV4 interaction, whereas PACSIN1 loses its binding and consequently TRPV4 regulation
ability in the presence of the neuropathy-causing TRPV4 R269C mutation. Expression lev-
els of the PACSIN chimeras in HEK293T cells were significantly lower compared to wild-type
PACSIN1-3 (see figure 4.16 B), which could also be a reason for the missing co-IP of any
of the PACSIN chimeras with TRPV4. Low-affinity or transient PPIs may not be detected in
co-IPs, as extensive washing and mechanical/chemical stresses during the experiment could
118
Chapter 4. Results
disrupt the PPI of the target complex. Therefore it would still be possible that the chimera PAC-
SIN2+3 still directly binds to TRPV4 via its SH3 domain in cellulo, leading to a TRPV4 regulation.
This would indicate that already low amounts of a TRPV4-regulating PACSIN isoforms/chimera
are sufficient to achieve TRPV4-mediated Ca2+-influx dampening and also explain the simi-
lar Ca2+-influx dampening effect of PACSIN1 and PACSIN3 (figure 4.16 A) even though the
TRPV4-PACSIN1 interaction seems to be weaker in the context of HEK293T cells (figure 4.16
B).
119
Chapter 4. Results
4.4 Small but powerful - the small GTPase RhoA interacts with the
TRPV4 Ankyrin Repeat Domain
Parts of this chapter were published in: McCray, B.A., Diehl, E. et al. Neuropathy-causing TRPV4 muta-
tions disrupt TRPV4-RhoA interactions and impair neurite extension. Nat Commun 12, 1444 (2021).69
The author of the present thesis (Erika Diehl) contributed purification, characterization and nuclear mag-
netic resonance (NMR) spectroscopy-based PPI studies between recombinantly expressed 15N-RhoA
and hsV4 ARD wt as well as hsV4 ARD R269C.
TRPV4 modulates cell morphology and stiffness via cytoskeleton components, namely mi-
crotubules and actin scaffolds.69,161–165 A generic bottom-up proteomics approach, conducted
by MD (RG , Department of Neurology, Johns Hopkins University
School of Medicine) with immunopurified human TRPV4-FLAG overexpressed in HEK293T
cells indicated the small GTPase RhoA as a putative direct TRPV4 interactor.69 These data
complemented the recently published TRPV4-RhoA interaction identified via a Y2H screen.136
RhoA itself is a pivotal regulator of the actin cytoskeleton, inducing stress fiber formation, for
example.166–168 Therefore a possible direct PPI and actin regulating interplay between TRPV4
and RhoA was investigated and if these putative interactions were aberrant in the presence of
disease-causing mutations of TRPV4. MD could show with comprehensive
co-IP studies in HEK293T and MN-1 (a murine cholinergic motor neuron cell line) cells, that
TRPV4 pulls down RhoA and vice versa. Furthermore, co-IPs of MN-1 cells transiently trans-
fected with TRPV4 and inactive RhoA-GFP (T19N), or constitutively active RhoA-GFP (Q63L)
demonstrated that TRPV4 interacts with inactive, GDP-bound RhoA T19N, but not active, GTP-
Figure 4.19 (facing page): Purification of hsV4 ARD wt, hsV4 ARD R269C and 15N-isotope labeled human RhoA (15N-RhoA
). A Schematic topology model of hsV4 ARD wt, hsV4 ARD R269C and 15N-RhoA used for NMR spectroscopy measurements.
His6 = hexa-histidine tag (single letter code: HHHHHH), TEV = Tobacco Etch Virus Cleavage Site (single letter code: ENLYFQG).
His6-tags with preceding or subsequent TEV Cleveage Site were removed. B SEC purifications of 15N-RhoA, hsV4 ARD wt and
R269C. SEC runs were performed with a HiLoad Superdex 75 pg preparative SEC column. Inlets show Coomassie stained 15 %
SDS-PAGEs of collected and concentrated fractions after SEC. x-axis: eluted volume in mL, y-axis: absorbance at 280 nm, M =
marker. C For 15N-RhoA, the intrinsic GTPase activity was determined via the luminescence-based GTPase-Glo Assay. GTPase
activity is inversely correlated to measured luminescence intensity. A paired two-tailed t-test with 3 independent measurements
including 4 technical replicates was performed, ***p < 0.001. Error bars indicate SD. D + E Far UV CD spectra of purified hsV4
ARD wt and hsV4 ARD R269C as well as 15N-RhoA, respectively. Spectra were measured at 293 K and proteins were used at
1µM concentration in RhoA SEC buffer with a final concentration of 20 mM NaCl. F Secondary structure prediction of shown far
UV CD spectra shown in subfigures D and E, performed with CAPITO.93 The predicted α-helix/β-sheet/random coil content of the
purified proteins is for hsV4 ARD wt 78 %/0 %/22 %, hsV4 ARD R269C 68 %/0 %/32 % and 15N-RhoA 27 %/24 %/49 %. y-axis:
mean residue ellipticity ([Θ]mrw,λ) in 10−3 deg cm2 dmol−1, x-axis: wavelength λ in nm.
120
Chapter 4. Results
A B
350 15 MW
N-hsRhoA [kDa] Coomassie
hsV4 ARD wt
300 hsV4 ARD R269C
75
1 193
250 50
hsRhoA N His6 TEV RhoA C
37
200
149 398
25
hsV4 ARD wt N ARD His C 150 A t 9C6
Rh
o w
15 26R
R269C
10
hsV4 ARD R269C N ARD His C 1006
hsV4 ARD
50
Superdex 75
0
50 60 70 80 90 100
40
C D Volume [mL]
60 25
15
hsV4 ARD wt N-hsRhoA
hsV4 ARD R269C 20
40 15
10
20 5
0
0 -5
-10
-20 -15
-20
-40 -25
200 210 220 230 240 250 260 200 210 220 230 240 250 260
l [nm] l [nm]
E F
*** 1 .0 random coil
120000
b-sheet
0 .9 a-helix
0 .8
100000
0 .7
80000 0 .6
0 .5
60000
0 .4
0 .3
40000
0 .2
0 .1
20000
0 .0
hs- w
t
9C
5
0 1 N oA 2
6
l s h R
nt
ro h R
15
o N
-
oA hsV4 ARDC Rh
121
3 2 -1
q [10 deg cm dmol ]
mrw, l
Luminescence [RLU]
3 2 -1
q [10 deg cm dmol ] Abs [mAu]
mrw, l 280nm
sec. structure content
Chapter 4. Results
bound RhoA Q63L.169 These results were further confirmed with Co-IPs in the presence of ei-
ther excess GDP or an unhydrolyzable GTP analog (GTPγS) to favor the inactive or active form
of RhoA. Additionally, co-IPs with immunopurified RhoA-myc and recombinantly expressed,
purified human TRPV4 N-terminus (hsV4N) and ARD (hsV4 ARD), respectively, indicated a
direct interaction between these proteins. Further co-IPs in transiently transfected MN-1 cells
showed an abolished TRPV4-RhoA PPI in the presence of neuropathy-causing TRPV4 mutants
(R232C, R237L, R269C, R315W), but not skeletal dyplasia-causing mutations (I331F, D333G).
These neuropathy-mutant PPI disruptions were shown to be TRPV4-channel activity indepen-
dent, as neither the TRPV4 anatgonist HC-067047 (HC-067) nor the pore-blocking M680K mu-
tation restored the PPI between RhoA and TRPV4 harboring respective neuropathy-causing
mutations.
15
I80 E47 N-RhoA15
N-RhoA + hsV4 ARD wt
15
15
N-RhoA + hsV4 ARD R269C N d
[ppm]
L57 V139 R150
K6
V48 I95 A3
D59
D49 E172 Y42
L81 E40
A2
V53 I10
V9
V35
E47 V43
I113 E54M82
W58 I80
V35
V115
L55
F30 R5
E54
9 8
W58 M82
L57 V139 R150
K6
V48 I95 A3
D59
D49 E172 Y42
L81 E40
A2
V53 I10
V9
E47 V43
I113 E54M82
V35
W58 I80
V115
L55
F30 R5
1
9 8 H d [ppm]
1
9 8 H d [ppm]
Figure 4.20: NMR experiments revealed an decreased PPI between 15N-RhoA and the neuropathy-causing mutation R269C
in hsV4 ARD. Left: Representative 1D projection of 1H resonances from 2D 1H-15N NMR spectra of 15N-RhoA (grey) and in the
presence of either hsV4 ARD (blue) or hsV4 ARD R269C (red). Stronger signal decreases for 15N-RhoA in the presence of hsV4
ARD versus neuropathy mutant R269C indicate a decreased interaction between 15N-RhoA and mutant ARD. Right: Sections of
2D 1H-15N-NMR HSQC spectra of 15N-RhoA: Overlay of spectra of 1H-15N-RhoA 2D HSCQs of 15N-RhoA on its own (grey) in
the presence of hsV4 ARD (blue, upper right) show line broadening, indicating the formation of a high molecular weight complex.
In contrast, addition of hsV4 ARD R269C (red, lower right) shows only minor effects on the 15N-RhoA spectrum. 15N-RhoA
backbone NMR assignments were transferred from previously published data170. NMR spectra were recorded on a Bruker 600
MHz spectrometer equipped with a cryogenic triple probe at 298.15 K and processed using Bruker TopSpin 3.2.
122
0 5 [rel] 10
128 125 128 125
Chapter 4. Results
Due to the clustering of investigated neuropathy-causing TRPV4 mutations in the TRPV4 ARD
(hsV4 ARD), the author of this thesis examined the direct PPI between recombinantly expressed
and purified 15N-RhoA and wild-type hsV4 ARD (hsV4 ARD wt) as well as hsV4 ARD R269C
neuropathy mutant (hsV4 ARD R269C) via nuclear magnetic resonance (NMR) spectroscopy.
To characterize this PPI in detail, 1H,15N NMR measurements of RhoA in the presence of hsV4
ARD and hsV4 ARD harboring the neuropathy-causing mutation R269C (hsV4 ARD R269C)
were performed. For this, recombinantly expressed hsV4 ARD and hsV4 ARD R269C, as
well as 15N-labeled human GDP-bound RhoA (hereafter referred to as 15N-RhoA) were puri-
fied (see subsection 3.7.5). Recombinant proteins showed the expected molecular weights via
SDS-PAGE with subsequent Coomassie-staining (figure 4.19 A inlet) and secondary structures
determined by CD spectroscopy (figure 4.19 D and E). Additionally, 15N-RhoA showed enzy-
matic functionality in a luminescence-based GTPase assay (figure 4.19 F). After confirmation
of the protein integrity, two-dimensional 1H,15N-NMR spectra of 15N-RhoA were recorded (see
section 6.6 figures 6.4 and 6.5 for full spectra). The obtained spectra agreed with previously
published NMR data (BMRB:16668)170 and allowed a backbone resonance assignment trans-
fer of 93.6 %.
NBS Switch 1 NBS Switch 2 NBS hsV4 ARD wt hsV4 ARD R269C
1,2
1
0,8
0,6 2 s
0,4
0,2
0
-0,2
-0,4
-0,6 2 s
-0,8
-1
-1,2
# residues
Figure 4.21: Relative signal intensity changes of RhoA in the presence of unlabeled hsV4 ARD wt and R269C, respectively.
The largest decrease in signal intensity accounted for the E54 residue in 15N-RhoA, and a smaller decrease in E47, indicated by
arrow heads. NBS = Nucleotide binding site, Switch 1 and 2 = Switch 1 and 2 loop, 2σ = two SDs
To map the hsV4 ARD binding site on 15N-RhoA, spectra were recorded in the presence of
either hsV4 ARD or hsV4 ARD R269C (see figure 4.20). Signal intensities of 15N-RhoA res-
onances decreased significantly in the presence of hsV4 ARD, indicating formation of a large
molecular complex between hsV4 ARD (29.3 kDa) and RhoA (21.8 kDa). Addition of hsV4 ARD
R269C did not lead to such strong signal intensity decreases, indicating a reduced affinity for
15N-RhoA. To identify the 15N-RhoA residues that are strongly affected by the interaction with
hsV4 ARD, the relative signal intensity for every residue in the 15N-RhoA NMR spectra were de-
termined (figure 4.21). In total, peaks corresponding to 20 residues in 15N-RhoA exhibited peak
123
Norm. integral changes
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
57
59
61
63
65
67
69
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
101
103
105
107
109
111
113
115
117
119
121
123
125
127
129
131
133
135
137
139
141
143
145
147
149
151
153
155
157
159
161
163
165
167
169
171
173
175
177
179
181
Chapter 4. Results
integral decreases greater than two standard deviations (2σ) in the presence of hsV4 ARD,
whereas only two residues showed this decrease with hsV4 ARD R269C. This is also reflected
in the mean relative signal intensity change of 15N-RhoA in the presence of hsV4 ARD versus
hsV4 ARD R269C (figure 4.22, right).
Mapping the affected residues onto the crystal structure of GDP-bound RhoA (PDB: 1FTN)171
revealed that the interswitch and switch II regions of 15N-RhoA are primarily involved in hsV4
ARD binding (figure 4.22, left). The switch regions of RhoA are important regulatory sites
within the small GTPase which are sensors whether RhoA is in its GDP or GTP-bound state by
changing their conformational state upon the presence of hydrogen bonds to the γ-phosphate
in GTP ("loading-spring" mechanism). Depending on the switch conformations, interaction of
downstream effectors like Rho GDP dissociation inhibitors (RhoGDIs) and Rho guanine nu-
cleotide exchange factors (RhoGEFs) are regulated.169 The largest decrease in signal intensity
accounted for the E54 residue in 15N-RhoA, and a smaller decrease in E47, suggesting these
residues might be particularly important in TRPV4 binding (see figure 4.21, indicated by arrow
heads). These glutamic acids are unique to RhoA and are not present in the related Rho
GTPases Rac1 and Cdc42,
0,25
15
N-RhoA + hsV4 ARD wt
15 consistent with the results
0,2 N-RhoA + hsV4 ARD R269C
0,15
L72 demonstrating specificity
0,1
S73 Y74
C for interaction of TRPV4
0,05
L8
A61 K6 R5
D59 0 with RhoA and not Rac1V139 W58
L57 L55 N
E54 -0,05
E40 V53 or Cdc42.69 Mutation of
ADP
V43 -0,1
L22 A44
F25 E54 to A completely abol-
-0,15
PDB: 1FTN -0,2 ished the interaction of
-0,25
RhoA with TRPV4 in co-
Figure 4.22: Interaction sites of hsV4 ARD with 15N-RhoA. Left : NMR signal intensities IPs conducted by MD
in the presence of hsV4 ARD mapped on a crystal structure of GDP-bound human RhoA
(PDB: 1FTN)171. Residues with significant decreases in signal intensity (indicated in dark Brett A. McCray, sug-
purple) are predominantly localized in the RhoA switch regions. White parts correspond to gesting that E54 may be
residues for which no peaks could be identified in the measured 1H-15N 2D HSQC spectra.
important for electrostatic
Right : Average integral change of 15N-RhoA residues in the presence of hsV4 ARD or hsV4
ARD R269C. Error bars indicate SEM. interaction with positively
charged arginine residues
within the hsV4 ARD that appear to be critical for RhoA binding.69 Both mutants, E47 and E54,
showed preserved interaction with RhoGDI, indicating that they were properly folded and func-
tional. Our observations thus provide a potential structural explanation for the disruption of
RhoA interaction by neuropathogenic mutations within hsV4 ARD. MD Brett A. McCray showed
124
Mean integral change
Chapter 4. Results
via live Ca2+-influx assays and co-IPs with transiently transfected MN-1 cells, that RhoA bind-
ing results in suppression of TRPV4 channel activity but also to RhoA inhibition. Activation of
TRPV4 with the potent TRPV4 agonist GSK-101 in transfected HEK293T cells lead to RhoA
activation and cytoskeletal changes. The regulation of cell morphology by an interplay between
TRPV4 and RhoA was furthermore investigated by neurite-like outgrowth length quantification
of MN-1 cells. MN-1 cells transfected with TRPV4 alone showed pronounced neurite-like out-
growth, whereas the neuro-pathy-causing R237L mutation failed to promote neurite-like out-
growth in MN-1 cells. Co-expression of RhoA with TRPV4 therefore strongly inhibited this
TRPV4-mediated neurite-like outgrowth, which was rescued by RhoA inhibition of the potent
and specific RhoA inhibitor exoenzyme C3 transferase. Strikingly, also the neuropathy-causing
mutation TRPV4 R237L phenotype could be partially rescued via RhoA inhibition, indicating
that elevated RhoA activity due to interrupted RhoA-TRPV4 interaction contributes to patholog-
ical neurite-growth outcomes in TRPV4-caused neuropathies. This hypothesis was tested in
vivo with a D. melanogaster model where it was found that expression of the Drosophila RhoA
ortholog Rho1 by expression of dominant negative Rho1 T19N rescued neuronal degeneration
with expression of neuropathy mutant TRPV4 R269C.
Within this cooperation, RhoA was identified as a direct interactor of TRPV4 with its ankyrin
repeat domain, a notorious hot-spot for disease-causing mutations in TRPV4. RhoA was in-
dentified as a mediator of TRPV4-induced cell structure changes and the here described find-
ings strongly hint towards that disruption of TRPV4-RhoA binding is one determinant of tissue-
specific toxicity of TRPV4 neuropathy mutations.
125
Chapter 4. Results
4.5 All or nothing - interaction of ITCH requires the full TRPV4
N-terminus in vitro
Ubiquitination and deubiquitination of transmembrane proteins are key mechanisms in regulat-
ing their surface expression. Wegierski et al. showed that ITCH-mediated TRPV4 ubiquitina-
tion does not increase TRPV4 degradation but decreases the TRPV4 amount in the plasma
membrane.172 It was shown that this ubiquitination took place at the TRPV4 N-terminus and not
the C-terminus by overexpressing the respective FLAG-tagged cytosolic TRPV4 domain along
with ITCH in HEK293T cells with subsequent western blotting of the cell lysates against FLAG
and ubiquitin. As ITCH harbors four WW domains which are hypothetically able to interact with
the TRPV4 PRR, these results suggested a direct interaction between ITCH and TRPV4, even
though the cell lysate could have provided additionally required adaptor proteins for a proper
PPI between ITCH and the TRPV4 N-terminus. Shukla et al. showed a β-arrestin 1 mediated
ubiquitination and functional down-regulation of TRPV4 via an angiotensin I mediated AT1aR
activation in HEK293T cells, indicating that β-arrestin 1 is required for a GPCR-regulated ITCH-
mediated TRPV4 ubiquitination.173
To investigate a possible direct interaction between ITCH and the human TRPV4 N-terminus
(hsV4N) in vitro, recombinantly expressed human full-length ITCH was successfully purified
for the first time to date (see figure 4.23 and subsection 3.7.7). To distinguish possible mini-
mal binding sites within hsV4N, hsV4N ARD and hsV4N ARD with the PRR (hsV4 ARD-PRR)
were also recombinantly expressed and purified (see subsection 3.7.1 and 3.7.2). All proteins
showed expected molecular weights and secondary structures determined by CD spectroscopy
Figure 4.23 (facing page): Purification of recombinant TRPV4 N-terminal constructs and human ITCH. A Topology model of
recombinantly expressed TRPV4 N-terminal constructs and human ITCH. His6 = hexa-histidine tag (single letter code: HHHHHH),
TEV = Tobacco Etch Virus Cleavage Site (single letter code: ENLYFQG), C2 = C2 domain, WW1+2 = WW domains 1 and 2,
WW3+2 = WW domains 3 and 4, HECT = homologous to E6-AP C-terminus domain. His6-tags with preceding or subsequent TEV
Cleveage Site were removed. B SEC purification of full-length human ITCH. C SEC purifications of TRPV4 N-terminal constructs
and ITCH. SEC runs were performed with either a HiLoad Superdex 200 or a HiLoad Superdex 75 pg preparative SEC column.
Inlets show Coomassie stained 15 % SDS-PAGEs of collected and concentrated fractions after SEC. x-axis: eluted volume in
mL, y-axis: absorbance at 280 nm, M = marker. D Far UV CD spectrum of full-length human ITCH. E Comparison of far UV CD
spectra of purified TRPV4 N-terminal constructs. Spectra were measured at 293 K and proteins were used at 1µM concentration
in either SEC Buffer with a final NaCl concentration of 30 mM (TRPV4 N-terminal constructs) or ITCH SEC buffer with a final
concentration of 20 mM NaCl (ITCH). Inlets show secondary structure prediction performed with CAPITO.93 The predicted α-
helix/β-sheet/random coil content of the purified proteins is for hsV4N 53 %/0 %/.47 %,hsV4 ARD-PRR 60 %/0 %/ 40 %, hsV4
ARD 68 %/0 %/32 % and ITCH 29 %/20 %/51 %. x-axis: mean residue ellipticity ([Θ]mrw,λ) in 10−3 deg cm2 dmol−1, y-axis:
wavelength λ in nm.
126
Chapter 4. Results
A 1 5 99 326 391 438 511 569 903
ITCH N His6 TEV
WW WW
C2 HECT
1+2 3+4 C
1 136 146 149 398
hsV4N N PBD PRR ARD His6 C
hsV4 ARD-PRR N PRR ARD His6 C
hsV4 ARD N ARD His6 C
B C
350 350 hsV4 ARD-PRR M
ITCH M
[kDa] Coomassie hsV4 ARD Coomassie [kDa]
250 150
300 150
300 100
75
100
75
ITCH 250
50
250
50 37
200
200 37
25
150
150 25 Superdex75
15
15
100
100
4NV sV
4
RR sV
4
hs h -P h RD
Superdex200 50
AR
D A
50
0
0
40 50 60 70 80 90 100
40 50 60 70 80 90 100
Volume [mL]
Volume [mL]
D E
1 .0 1 .0random coil 60 random coil
140 b-sheet hsV4N b-sheetITCH 0 .9 0 .9a-helix
0 .8 hsV4 ARD-PRR
a-helix
0 .8
120
0 .7 hsV4 ARD 0 .7
100 0 .6 0 .6
40
0 .5 0 .5
80
0 .4 0 .4
60 0 .3 0 .3
0 .2 0 .2
40
0 .1 20 0 .1
20 0 .0 0 .0
TC
H N 4
I V4 sV
4
RR
V
s h -P h
s D
0 h D AR
AR
-20
0
-40
-60
-80
-20
-100
-120
-140 -40
200 210 220 230 240 250 260 200 210 220 230 240 250 260
l [nm] l [nm]
127
Abs [mAu]
280nm
3 2 -1
q [10 deg cm dmol ]
mrw, l
sec. structure content
q [10 deg cm dmol ] Abs [mAu]
mrw, l 280nm
sec. structure content
Chapter 4. Results
(see figures 4.23 A-E). The topology model in figure 4.23 A shows the domains full-length ITCH
is harboring. Besides the already mentioned WW-domains, ITCH consists of a C2 and catalyti-
cally active HECT (homologous to E6-AP C-terminus) domain. C2 domains bind phospholipids
for (plasma) membrane localization, either in Ca2+ dependent or Ca2+ independent manner,
depending on the C2 domain family.174 The catalytic HECT domain interacts with E2 ligases
to obtain ubqiquitin via trans-thioesterification and ultimately carries out the ubiquitination of
substrate proteins.174,175 Zhu et al. provided the until to date only near-full length structure of
ITCH via X-ray crystallography. However, the murine construct is lacking the N-terminal C2
domain (∆C2-ITCH) and WW3+4 are not resolved (PDB:5XMC).175 Nevertheless, this murine
∆C2-ITCH structure is consistent with individual resolved structures of WW1+2 and HECT do-
mains. The structure of Zhu et al., combined with several resolved structures of the isolated
domains, revealed an overall α-helical fold for the HECT domain, whereas the C2 and WW do-
mains exhibit β-sheet folds.175–177 Enzymatic activity of purified ITCH was shown in an in vitro-
ubiquitination assay (figure 4.25 A). The putative complex formation between ITCH and respec-
tive TRPV4 N-terminal construct was probed via Blue-native PAGE (BN-PAGE). For hsV4N a
complex at approx.150 kDa could be observed, which corresponds to an ITCH (approx.100 kDa)-
hsV4N (approx.50 kDa) complex with a stoichiometry of 1:1. To confirm this, respective band
was cut out (figure 4.24 B, red square) and mass spectrometric measurements were carried
out (Johns Hopkins Mass Spectrometry and Proteomics Facility), confirming the presence of
both proteins in indicated band. These results show that ITCH physically interacts with hsV4N,
but not with hsV4 ARD and hsV4 ARD-PRR. Cross-link mass spectrometry (XL-MS) measure-
ments with hsV4N and ITCH confirmed this direct PPI and also hint towards the requirement of
full hsV4N for the interaction with ITCH (figure 4.24 C). Surprisingly, no lysine residues between
hsV4N and the ITCH HECT domain were cross-linked. The ITCH HECT domain harbors the
enzymatic activity site to receive ubiquitin from E2 ligases and to transfer this ubiquitin via trans-
thioesterification to the target protein.178,179 Also, no cross-links between the hsV4N PRR and
ITCH were detected, underlining the results of shown BN-PAGE results, that the full hsV4N is
involved in the PPI (figure 4.24). To determine if ITCH is also able to ubiquitinate hsV4N in vitro,
an ubiquitination assay with subsequent SDS-PAGE and Coomassie staining was performed
with ITCH and respective hsV4N constructs (figure 4.25).
128
Chapter 4. Results
A B
+ +
c LA
G G
yc c
m LA
G A y AGc L R RRy
m 4-
F m R
H- -F
y -Fm 4 H- -F
L P -P
H-
-
V C 4 - V C 4 D D + + D + D S
T
P
TC R I
T PV M TC
H
RP IT P
V R AR H H ARA H R G
I T TR [kDa] I T R 4
N 4 M C N C 4 A +T CH sV sV
4 V ST
T
s I sV
4 IT C
T h h h G [kDa] h hs
V IT V4 H
I hs
C
IT
100
75 250
100
150
75 100
100 50
75
100
75
Input (5 %) co-IP (a-myc)
Pure proteins Protein mixtures
C
1 5 99 326 391 438 511 569 903
WW WW
ITCH N C2 HECT1+2 3+4 C
1 149 398 = lysine residue
hsV4N N PBD PRR ARD His6 C
Figure 4.24: ITCH and hsV4N are direct interaction partners. A α-myc immunoprecipitation of ITCH-myc shows
co-immunoprecipitation of TRPV4-FLAG co-transfected in HEK293T cells and confirms ITCH as a part of the TRPV4
interactome172,173B Blue-native PAGE with purified TRPV4 N-terminal constructs and ITCH (see figure 4.23) shows complex
formation at approx. 150 kDa (red square) between ITCH and hsV4N, confirmed by mass-spectrometric measurements.C Cross-
link mass spectrometry (XL-MS) confirms a direct interaction between ITCH and hsV4N. Green bars indicate native lysine residues,
whereas lysine residues involved in DSS-mediated cross-linking are additionally numbered. Black lines show cross-links between
respective lysine residues. XL-MS measurements were kindly carried out by M.Sc. ,
University of Konstanz)
At time point 60 min a band of approx.10 kDa (figure 4.25 A, red square) above the unmodified
hsV4N band was observed, indicating an ubiquitination as ubiquitin has a molecular weight of
8.6 kDa. For better visualization, the time point 90 min is also shown. hsV4 ARD and hsV4
ARD-PRR showed no higher molecular weight bands and therefore no ubiquitination. For sub-
sequent mass spectrometric identification of hsV4N ubiquitinaton sites, the red squared band
after 60 min was cut out to rule out time-dependent unspecific ubiquitinations, as this was the
earliest time point with a visible band for modified hsV4N. Four lysine residues in the intrinsi-
cally disordered region (IDR) of hsV4N were identified as ubiquitination sites: K77, K101, K130
and K136. The IDR alone includes 11 lysine residues and the whole hsV4N a total of 23 ly-
sine residues, depicting a high lysine specificity of ITCH-mediated ubiquitination. As mentioned
above, ubiquitin has a molecular weight of 8.6 kDa.
129
34
70
211
a-myc, a-myc, a-FLAG, a-FLAG,
2 min 30 sec 2 min 30 sec
251
350
478
529
555
Chapter 4. Results
A
RR RR R
R
RR RR
D D-
P P  P P P
R R RD D
- D D- D - -
A A A AR AR AR AR AR
D RD RD
4 4 4N 4 4 4N 4 4 4N 4 4 4N M
A A N
sV V V
4 4 4
h hs hs hs
V sV V V V V V V Vh hs hs hs hs hs hs hs [kDa] hs
V V
hs hs
V
100
ITCH
75
50
37
25
20
15
Ubiquitin 10
t [min] 0 15 30 60 90
B
C
N
K77
K101
PRR PBD
K136 K130
ARD
PDB: 4DX1 = ubiquitinated lysine residue
= non-ubiquitinated lysine residue
Figure 4.25: ITCH ubiquitinates hsV4N directly. A In vitro ubiquitination assay shows ubiquitination of hsV4N by ITCH, indicated
by the bands in red squares at time points 60 min and 90 min. The band at time point 60 min was cut out and supplied to mass
spectrometric detection for ubiquitination (Johns Hopkins Mass Spectrometry and Proteomics Facility). See subsection 3.10.3 for
a protocol of the assay. B Mass spectrometry revealed the direct ubiquitination of four lysine residues (K77, K101, K130 and K136)
in the hsV4 IDR by ITCH, indicated by green bars.
Therefore, hsV4N is present in a monoubiquitinated state in this assay, where each of above
mentioned lysine residues is ubiquitinated once, respectively. The detected relative abundances
of respective monoubiquitinated lysine harboring peptides decrease from K101>K77>K136>K130
(see section 6.7). Monoubiquination does not target proteins for proteosomal degradation,
but rather serves as a signal for membrane trafficking and endocytosis.172,180,181 These find-
ings also underline the results of Wegierski et al., who determined TRPV4 to be rather multi-
than polyubiquitinated.172 Furthermore it was shown that the amount of internalized TRPV4 in-
creased in the presence of ITCH, suggesting an increased ITCH-mediated TRPV4-endocytosis
via monoubiquitination.172,173 But why is there no HECT-hsV4N IDR interaction in the shown
XL-MS data in figure 4.24 C?
130
Chapter 4. Results
A
1 5 99 326 359 391 419 438 511 569 903
WW WW WW WW
hsITCH N C2 HECT1 2 2L 3+4 C
= lysine residue
B
WW2
HECT
mm K311
hs K350
WW2L mm C832
hs C871
N
PDB: 5XMC
Figure 4.26: Cross-link mass spectrometry (XL-MS) measurements showing inter- and/or intramolecular interactions be-
tween ITCH molecules. A Topology model of full-length human ITCH with respective inter- and/or intramolecular self-crosslinks of
ITCH. Green bars indicate native lysine residues, whereas lysine residues involved in DSS-mediated cross-linking are additionally
numbered. Dark arches show cross-links between respective lysine residues. XL-MS measurements were kindly carried out by M.
Sc. Jasmin Jansen (Working group Stengel, University of Konstanz).B Crystal structure of autoinhibited mouse ITCH (mmITCH)
(PDB:5XMC). For better comparison with the used human constructs in this theses, highlighted residues are indicated for mouse
(mm) with the corresponding human (hs) residue below. lysine residues are indicated as light green spheres. Mouse cysteine 832
(mm C832) is shown as yellow sphere, indicating the position of the catalytic active site for ubiquitin transthiolation in the HECT
domain. The structure here is lacking the C2 domain, furthermore the WW3 and 4 domains were not resolved. The only structurally
resolved lysine in shown murinal structure which is involved in cross-links shown in A mouse lysine 311 (mm K311),which is the
corresponding lysine for human lysine 350 (hs K350).175
It is known that HECT E3 ligases like ITCH exist in an autoinhibited state.175,179 In this au-
toinhibited state, the WW2 domain and a part of the linker region connecting WW2 and WW3
(referred as WW2L) bind to a hydrophobic surface opposite to the catalytic active site of the
ITCH HECT domain, therefore allosterically locking the HECT domain in an inactive state (fig-
ure 4.26 A). However, binding of the ITCH interaction partner Ndfip1 to the ITCH WW2 domain
releases ITCH out of its autoinhibited state.175 The XL-MS data thus could represent the initi-
ation step of the ITCH-hsV4N interaction, where hsV4N releases ITCH out of its autoinhibitory
state and ITCH would be ready to receive an ubiquitin from an E2 ligase in its HECT domain.
The presence of ITCH in this autoinhibited state could not be determined via self-link (inter- or
intramolecular between ITCH) XL-MS data (figure 4.26), as the WW2 domain and WW2L does
not harbor any lysine residues which could have been cross-linked to lysine residues in the
131
56
211
249
350
478
513
251
Chapter 4. Results
HECT domain. Furthermore, neither in intermolecular cross-links between hsV4N and ITCH
(figure 4.24 C) nor in self-linked cross-links between or within ITCH (see 4.26 B) any HECT
lysine participated in cross-linking. This could be due to the location of most HECT lysine
residues, as they are buried in the N- and C-lobe of the HECT domain and therefore are un-
reachable for DSS for cross-linking (figure 4.26 B). Ubiquitin binding itself leads to dramatic
conformational changes in the HECT domain. HECT domains consists of a so-called N- and
C-lobe, with the C-lobe harboring the catalytically active site. These lobes change their overall
orientation from a "closed" T-shape to an "open" L-shape upon ubiquitin binding, enabled via a
flexible hinge region between the two lobes. Therefore the data suggests, that hsV4N probably
releases ITCH from its autoinhibited state, enabling ubiquitin binding to the HECT domain via
an E2 ligase. Due to the ubiquitin-binding, the HECT domain changes from a "closed" T-shape
to an "open" L-shape, enabling an interaction of the hsV4N IDR with the HECT domain for IDR
ubiquitination.182
Interestingly, two of the ubiquitinated TRPV4 lysine residues are in close proximity to the pro-
line rich region (PRR, K136) and the phosphoinositide-binding domain (PBD, K130) of hsV4N,
two important regulatory sites in TRPV4. These modifications could thus lead to an altered
binding behavior between hsV4N and other proteins, for example, between PACSIN3 and the
TRPV4 PRR. As already described in section 4.3, co-expression of PACSIN 1 and 3, respec-
tively, with TRPV4 lead to a dampened hypoosmolarity induced TRPV4-mediated Ca2+-influx
in HEK293T cells (figure 4.16). Furthermore, it was shown that the presence of PACSIN3 in-
creases the plasma membrane amount of TRPV4 by probably inhibiting endocytosis, whereas
ITCH decreases the TRPV4 amount in the plasma membrane via endocytosis.70,172 Here, two
different regulating mechanisms lead to a decreased basal TRPV4 activity. The ubiquitination
close to the TRPV4 PRR thus could therefore be a regulating switch between these two mech-
anisms: whereas PACSIN3 could provide a transient and fast TRPV4 desensitization, ITCH
could disrupt this interaction via the K130 and K136 monoubiquitinations, therefore sterically
hindering the PACSIN3 SH3 domain to interact with the TRPV4 PRR. This then could lead to
a TRPV4 endocytosis, serving a TRPV4 "inactivation" for a longer time range.172 Furthermore,
the monoubiquination of K136 next to the PBD could lead to an impaired TRPV4-PIP2 interac-
tion, resulting in an altered TRPV4 regulation. Opposite consequences for the TRPV4 activity
were shown upon PIP2 binding, pointing out the need for further investigations of the conse-
quences of TRPV4-lipid interactions.39,183,184
Goretzki et al. showed via elaborate and comprehensive biophysical studies a structural and
dynamic coupling between the TRPV4 IDR and ARD.23 Consequently, comparably large post-
132
Chapter 4. Results
translational modifications (PTMs) in the TRPV4 IDR could thus lead to modified interactions
with the TRPV4 ARD with impacts on ARD-mediated PPIs, for example. The results shown here
therefore contribute to a further understanding of the PPI between hsV4N and ITCH.
133
Chapter 4. Results
4.6 TRPV4 and the actin cytoskeleton - connecting scientific
disciplines
In preceding MS studies of the hsV4N interactome, conducted in the authors master thesis,
a possible Ca2+-dependent interaction between smooth muscle α-actin and hsV4N was ob-
served, where α-actin amounts were higher in the samples with Ca2+-supplementation.76 This
findings were especially intriguing, as TRPV4 is known to influence the microtubule and actin cy-
toskeleton, with consequences for cell stiffness, migratory behavior and morphogenesis.185–187
Goswami et al. showed a direct interaction between the TRPV4 C-terminus and actin in co-
sedimentation assays, but left out hsV4N in these studies due to expression problems. There-
fore, Goswami et al. argue that hsV4N could also play an important regulatory role in the shown
colocalization between TRPV4-GFP and F-actin in transiently transfected F11 cells.185 This
possible cytoskeletal mediating role of hsV4N is underlined by the direct interaction between
hsV4 ARD and the small GTPase RhoA shown in this thesis (section 4.4). RhoA is a prominent
cytoskeleton regulator, playing a key role in stress fiber formation and actin polymerization.166,167,169.
As a starting point to elucidate the role of hsV4N in actin regulation, the potential Ca2+-dependent
colocalization between the actin cytoskeleton and TRPV4 was investigated with fluorescence
microscopy. HEK293 cells stably expressing human TRPV4-cGFP were treated either with
200 nM HC067 (TRPV4 antagonist) for 15 min at 37 °C, 30 nM GSK-101 for 1 min at 37 °C or
left at 37 °C for 15 min without any supplementation. F-actin was visualized by subsequent
phalloidin-TRITC staining (figure 4.27, for full immunostaining procedure see section 3.3).
Although plenty of colocalization programs are available, either as ImageJ plugins or as com-
mercial softwares, the algorithms and parameters of these programs are poorly described.188
Furthermore, most of the programs do not provide automated image analysis, making statistical
evaluations of colocalization quantification tedious and time-consuming. To circumvent the us-
age of "black box" programs and to provide large-scale image analysis, the theoretical chemist
B. Sc. joined this project to contribute an in-house software for colocaliza-
tion studies: ELSEXY, short for Efficient Large Scale Evaluation of cross(X)-correlation Yields.
Figure 4.27 shows representative fluorescence images of stably transfected HEK293 cells ex-
pressing hsTRPV4-cGFP after various treatments.
134
Chapter 4. Results
135
hsTRPV4-cGFP F-actin Merge DAPI
Figure 4.27: Fluorescence microscopy images reveal stress fiber formation upon TRPV4 activation in stably transfected HEK293 cells. Stress fiber formation was especially pronounced
upon treatment with 30 nM GSK-101 for 1 min at 37 °C, with clearly visible dorsal and ventral stress fibers as well as transverse arcs, whereas only temperature activation of TRPV4 at 37 °C
lead to a milder phenotype with some ventral stress fibers visible. Treatment with 200 nM of the TRPV4 antagonist HC-067 for 15 min at 37 °C showed no stress fiber development. F-actin was
visualized via phalloidin-TRITC staining (see section 3.3). Scale bar = 10µm. Images were taken with a Zeiss Axio Observer Z1 inverted widefield microscope.
HC-067 Temp. [37 °C] GSK-101
Chapter 4. Results
Upon chemical compound activation with the potent TRPV4 activator GSK1016790A (GSK-
101), cells showed explicit stress fiber formation, hinting towards a hsV4N involvement in
cellular actin reassembly, as hsV4 ARD was shown to directly interact with inactive GDP-
bound RhoA, the key regulator of stress fibers in cells (see section4.4).166,167,169 TRPV4 ac-
tivation activates RhoA, leading to a loss-of-interaction between TRPV4 and RhoA.69 Activated,
GTP-bound RhoA then mediates stress fiber formation by downstream interactors like ROCK
and mDIA1.166 The mild stress fiber phenotype of cells after TRPV4 temperature activation
hints towards a Ca2+-
GFP and TRITC
concentration dependency
GSK-101 MGFP
of stress fiber formation, GSK-101 MTRITC
as more physiological stim- HC-067 MGFP
uli lead to lower amount of HC-067 MTRITC
Ca2+-influx compared to Temp. MGFP
GSK-101 treatment (see Temp. MTRITC
also section 4.3, figure 0 20 40 60 80 100
%
4.16). To determine
2+ Figure 4.28: Calculated M-values of GFP (TRPV4-cGFP) and TRITC (F-actin stainedif TRPV4-mediated Ca with Phalloidin-TRITC) images show decreased TRPV4 and F-actin colocalization
influx leads to an in- upon TRPV4-mediated Ca2+-influx in HEK293 cells. For description of M-values see
equation 4.4 and corresponding text. Box plot whiskers indicate values inside 1.5-fold in-
creased TRPV4-actin colo-
terquartile range, green diamonds the arithmetic mean. Notches show 95 % confidence
calization, fluorescence im- intervall of mode. Outliers are shown as circles. nGSK-101=18, nHC-067=28, nTemp=33.
ages were subjected to
ELSEXY.
Shortly described, after hot-pixel and non-uniform background correction, ELSEXY calculates
the Pearson Colocalization Coefficient (PCC) r of subjected fluorescence images ( figure 6.8).
The PCC is a measure for the linear dependency of two measured values. To obtain the PCC
of the here obtained imgages, the measured values here are the fluorescence intensities of a
pair of corresponding pixels in two fluorescence microscopy images (IA and IB, see equation
4.3).189
= √ IA × IB − I × Ir A BA,B (4.3)
(I2A −
2 2
I 2A ) × (IB − IB )
Due to this linear dependency, the PCC not always perfectly describes the probable colocal-
ization in two images. Additionally, so-called M-values can improve the colocalization quan-
tification, by calculating the fraction of colocalized intensities. This is conducted by defining
thresholds for intensities. Pixel intensities above the defined thresholds in respective images
136
Chapter 4. Results
are considered as colocalized. These thresholds (TA, TB) result from a limit intensity (Ilim)
dependent evaluation of the PCC for pixels which are smaller than Ilim. M-values thereby
depend on the PCC r, as the thresholds depend on each other, shown in equation 4.4.189
∑
MA =
IA>T∑A∧IB>TB (4.4)
IA
with IB = m · IB + b.
Statistical evaluation of the colocalization between hsTRPV4-cGFP and F-actin via phalloidin-
TRITC with ELSEXY showed a decreased colocalization upon TRPV4-mediated Ca2+-influx
(figure 4.28 and also see figures 6.9 for negative controls). These results seem to op-
pose the above mentioned increased actin-levels detected in hsV4N MS samples upon Ca2+-
supplementation.76 One possible explanation could serve the sample preparation for subse-
quent MS experiments conducted by Diehl et al., which differ from the protocol conducted in
this work (section 3.8). Whereas in the present work a digitonin solution was used to release
cytosolic proteins into the supernatant of dish-cultured HEK293 cells, the previous protocol in-
cluded whole-cell lysis of HEK293 cells with a CHAPS-containing buffer. It was shown that
detergents like CHAPS lead to actin depolymerization from F-actin to G-actin, therefore hinting
towards the possibility that G-actin binds in a Ca2+-dependent manner to hsV4N.190 This hy-
pothesis is underlined by the results of Becker et al. who showed that the TRPV4 C-terminus
is also capable of binding G-actin in co-sedimentation assays.185 The possible interaction be-
tween G-actin and hsV4N could represent a regulatory loop between TRPV4, RhoA and F-actin.
TRPV4 activation leads to a reduced binding of RhoA to hsV4 ARD and activation of RhoA via
GTP binding.69 RhoA activation leads ultimately to stress fiber formation, while G-actin possibly
now interacts with hsV4N. In the course of this work it was tried to clone a TRPV4 construct lack-
ing the C-terminus to determine if only hsV4N is responsible for stress fiber formation via RhoA,
but until to this date, the cloning was not successful. However, above mentioned experimental
set-ups should be conducted to get a more detailed view on the hsV4N-actin interaction. Overall
it could be shown that different TRPV4 activators lead to Ca2+ concentration dependent stress
fiber formation in stably TRPV4-cGFP transfected HEK293 cells. Furthermore, the in house
software ELSEXY generated preliminary Ca2+-dependent colocalization data between TRPV4-
cGFP and F-actin. However, ELSEXY is still in the refinement process and will give raise to a
comprehensive, high-throughput colocalization studies in the future.
137
Chapter 5. Conclusion & Outlook
5 Conclusion & Outlook
Meeting at the hot-spot of disease causing TRPV4 mutations - the TRPV4 ARD
and its interaction partners DDX3X and RhoA
Inada et al. proposed altered tissue-specific protein-protein interactions (PPIs) as a reason for
the disease pathology and tissue specificity of respective TRPV4 mutations.27 First results in
this thesis underline before mentioned hypothesis by showing both, increased and abolished
PPIs between TRPV4 harboring neuropathy-causing mutations and several cytosolic proteins.
CMT2C-causing TRPV4 R232C showed increased interaction with DDX3X compared to TRPV4
wt in vitro and in cellulo, leading to decreased dxRNA-stimulated ATP-hydrolysis of DDX3X
upon this interaction in vitro (see sections 4.1 and 4.2). This altered PPI is particularly in-
teresting, as mutations leading to ATP-hydrolysis deficiency in DDX3X are connected to the
emergence of medulloblastoma, a rapidly growing and metastasizing form of infant cerebellum
tumor, and can be the cause of so-called X-linked intellectual development disorder Snijder
Broks type (MRXSSB, OMIM: 300958). MRXSSB patients show a spectrum of neuropathologi-
cal symptoms, but the most shared among patients are muscle hypotonia, movement disorders
and aberrant intellectual development.139,191–195 These symptoms, especially the intellectual
disability, hint towards a pivotal role of DDX3X in neuronal fetal development of the central ner-
vous system (CNS). DDX3X mutations were shown to affect CNS neurogenesis during fetal
development of the cerebral cortex. Additionally, it was shown that DDX3X plays a role in the in-
duction of the neuronal crest during fetal development, the progenitor structure from which the
peripheral nervous systems (PNS) develops from.196 TRPV4-associated neuropathies affect
the PNS, underlining the possible key role of the TRPV4-DDX3X interaction in PNS develop-
ment and maintenance and ultimately pathological consequences due to a TRPV4 mutation
induced aberrant TRPV4-DDX3X PPI, probably leading to stress granule (SG) hyperassembly
in the PNS.31,137,197
Pharmacological inhibition of ATP-hydrolysis or RNA-helicase activity in DDX3X via the small
138
Chapter 5. Conclusion & Outlook
molecules RK-33 or D16, respectively, showed diminished SG assembly upon arsenite induced
oxidative stress in UO2S cells. Already formed SGs upon stress treatment showed no altered
disassembly behavior upon RK-33 or D16 treatment, respectively.117 Especially regarding the
ATP hydrolysis inhibition, these findings seem to contradict the results of Valentin-Vega et al.
and in this thesis, indicating that impaired DDX3X ATP hydrolysis, either due to DDX3X mu-
tations or altered PPIs, spur SG assembly.119 Yang et al. confirmed direct binding of RK-33
to recombinant DDX3X (aa 1-508) with isothermal calorimetry (ITC) and proposed an impaired
RK-33 interaction via the active site of ATP hydrolysis due to the depletion of RK-33 binding
to DDX3X K230E in sedimentation velocity assays. This mutation resides within the DDX3X
Walker A motif, which is essential for DDX3X’s ability to hydrolyze ATP.198 However, the ex-
act binding mode of RK-33 to DDX3X still remains elusive and therefore the overall possible
structural consequences within DDX3X upon RK-33 binding. Thus, disease-causing mutations
which lead to impaired ATP hydrolysis in DDX3X still could be the cause of altered PPIs and
subsequent SG hyperassemblies, while pharmacological ATP hydrolysis inhibition influences
DDX3X in such a manner, that pathological SG-inducing PPIs are also impaired.
To shed more light in the consequences between the here shown elevated TRPV4 R232C-
DDX3X PPI, a multi-pronged approach should be performed. On one hand, NMR chemical
shift perturbation assays, corresponding to the experiments in section 4.4 with hsV4 ARD and
15N-labled RhoA, should be conducted to gain detailed insights in the direct interaction be-
tween DDX3X and hsV4 ARD and hsV4 ARD R232C, respectively. Furthermore, the influence
of RK-33 on possible structural changes in DDX3X could be also investigated via NMR and com-
pared to before mentioned NMR perturbation assays with hsV4 ARD and hsV4 ARD R232C to
reveal possible difference in ATP hydrolysis inhibition via a PPI or a pharmacological inhibi-
tion. Additional in vitro experiments should include further ATPase assays with ARDs of other
TRP channels or even other protein superfamilies to distinguish if this interaction exclusively
accounts for hsV4 ARD or also other ARDs. Furthermore, functional studies of DDX3X inter-
action for TRPV4 should be conducted in cellulo via Ca2+-influx assays as shown in 4.3. To
underline the hypothesis of SG hyperassembly due to aberrant TRPV4 R232C-DDX3X interac-
tion, in cellulo experiments with transfected cells should be conducted to investigate a TRPV4
(R232C)-mediated influence upon stress granule formation with DDX3X participation via im-
munofluorescence staining and subsequent microscopy, for example.
Additionally, the binary TRPV4 PPIs shown in this thesis should be expanded into ternary and
even quaternary PPI investigations. One starting point could be the possible ternary PPI be-
tween TRPV4, DDX3X and RhoA. For both proteins, a direct interaction with hsV4 ARD was
139
Chapter 5. Conclusion & Outlook
shown in this thesis. Furthermore it was shown that GSK-101 activation of TRPV4 wt in cel-
lulo leads to a decreased PPI between TRPV4 and RhoA or DDX3X, respectively, shown by
co-IPs.69,136 Furthermore, Phung et al. indicated a possible DDX3X-RhoA interplay by show-
ing elevated levels of GTP-bound (active) RhoA in human liver cancer cell lines (HT-144 and
A2058) upon DDX3X siRNA treatment, lowering but not depleting DDX3X levels in respective
cell lines.199 In the course of this thesis, upon the presence of a neuropathy-causing mutation,
DDX3X showed an increased TRPV4 PPI, whereas RhoA showed a decreased TRPV4 PPI.
It should be noted that two different neuropathy-causing mutations were used (TRPV4 R232C
with DDX3X and TRPV4 R269C with RhoA). Both mutations should be investigated in the con-
text of the ternary TRPV4-DDX3X-RhoA interaction.
Disorder matters - PPIs of the TRPV4 N-terminus underlines the pivotal role of
the intrinsically disordered region
Another possible ternary PPI which could be relevant in the context of neuropathy-causing
TRPV4 mutations includes TRPV4, PACSIN1 and ITCH. In this thesis it was shown that ITCH
directly interacts with only the full TRPV4 N-terminus (hsV4N), but not with the TRPV4 ankyrin
repeat domain (hsV4 ARD) or hsV4 ARD with the preceding proline rich region (hsV4 ARD-
PRR). Furthermore it was shown, that ITCH ubiquitinates four distinct lysines in the intrinsically
disordered region (IDR) of hsV4N (K77, K101, K130 and K136, see section 4.5). This is partic-
ularly interesting, as such post-translational modifications (PTMs) could interfere with possible
TRPV4 PPIs. With regard of the known interaction of PACSIN SH3 domains with the TRPV4
PRR (aa 136-144), ubiquitination of lysines K130 and K136 are especially interesting with re-
gard to possible disrupted PACSIN1/3 interactions with the TRPV4 PRR due to steric hindrance
after ubiquitination of respective lysine. But also, interaction of PACSIN1/3 could prevent TRPV4
IDR ubiquitination, resulting in two different regulating mechanism leading to a decreased basal
TRPV4 activity (see section 4.5). Desrochers et al. furthermore showed that the PACSIN1 SH3
domain interacts with the ITCH PRR located between the C2 and WW1 domain (aa 252 - 267).
With the shown loss-of-interaction between PACSIN1 and TRPV4 harboring the neuropathy-
causing R269C mutation with vanished TRPV4-mediated Ca2+-influx dampening by PACSIN1
as a consequence in cellulo (see section 4.3), this loss-of-interaction could also lead to an in-
creased interaction between PACSIN1 and ITCH in neurons. The physiological consequences
of this interaction still remain elusive, but one scenario could be an impaired interaction of ITCH
with other proteins - TRPV4 for example. Due to the location of the ITCH PRR between two other
140
Chapter 5. Conclusion & Outlook
important regulatory domains, namely the ITCH C2 domain and the ITCH WW1 domain, inter-
action with PACSIN1 could therefore impair possible interactions of at least these two domains,
hypothetically impairing plasma membrane localization via the ITCH C2 domain and other PPIs
of the ITCH WW1 domain, if not also of the other WW domains regarding the compact struc-
ture of ITCH in its autoinhibited state (shown in figure 4.26 B). As ITCH is known to mediate
TRPV4 endocytosis, a loss of interaction between PACSIN1 and TRPV4 R269C could there-
fore induce a vicious cycle where PACSIN1 and ITCH-mediated negative TRPV4 regulation are
both abolished. This would lead then to elevated neurotoxic Ca2+ levels due to the elevated
basal Ca2+ influx activity of this TRPV4 mutation (see also figure 4.17) in addition to the here
showed lost interaction between TRPV4 R269C and RhoA with the cytoskeletal consequences
(see 4.4).53,69
Neuropathy mutant
TRPV4
TRPV4
extracellular
plasma
membrane
intracellular
ARD ITCH ITCH
x
RhoA PR PR RhoA RR x
PACSIN1 PACSIN1
DDX3X DDX3X
N N
= increased interaction
= interacts with
x = abolished interaction
Figure 5.1: Tissue specificity of neuropathy-causing TRPV4 mutations occur due to aberrant tissue specific protein-
protein interactions Schematic representation of how TRPV4 neuropathy mutations lead to aberrant protein-protein interactions
investigated in the course of this thesis. It was shown that RhoA and DDX3X specifically interact with the a-helical TRPV4 ankyrin
repeat domain (ARD), whereas PACSIN1 is known to interact with the TRPV4 proline rich (PRR) region within the unstructured
intrinsically disordered region (IDR) and ITCH requires the full TRPV4 N-terminus, with a possible interaction with the TRPV4
PRR.22,69 In the presence of a neuropathy-causing TRPV4 mutation the interactions with RhoA and PACSIN1 are abolished, with
a possible consequence for the ITCH-TRPV4 interaction due to a putative increased PACSIN1-ITCH interaction. An increased
interaction between neuropathy mutant TRPV4 and DDX3X was also shown in this thesis.
To elucidate this, co-IPs with cells transfected with TRPV4 (R269C), ITCH and PACSIN1 should
be conducted to investigate the proposed binding competition between ITCH and PACSIN1 to
TRPV4. After getting details of the interaction between ITCH and TRPV4, either with NMR stud-
ies including the ITCH WW domains and a peptide including the TRPV4 PRR or via cryo-EM
with full length ITCH and hsV4N, latter experiment should be complemented with the TRPV4
141
Chapter 5. Conclusion & Outlook
R269C mutation. If both before mentioned methods should not be applicable, XL-MS studies
could be conducted including hsV4N R269C with ITCH and PACSIN1, respectively, to get a
detailed insight into the mutation-dependent altered PPIs.
With the comprehensive interactome study shown in this thesis, future investigations should
further validate the identified putative TRPV4 interactors shown in section 4.1, especially con-
sidering the proposed role of TRPV4 in cytoplasmic granule and/or stress granule formation via
its cytosolic N-terminus. Co-IPs with cells transfected with (mutated) TRPV4 and other here
detected prominent SG formation proteins like TIA-1 and IGF2BP2 (figure 4.6) should be con-
ducted, as well a possible TRPV4 mediation of altered stress granule formations under stress
conditions in cellulo.
142
Chapter 6. Appendix
6 Appendix
6.1 Appendix - Introduction
CUL1
PACS2 KIF3A KIF3B
EIF2AK3 STX5
RP2
HAVCR1 TNNI3
Ă-Trcp1 ID2
TNNI1 CBY1
HDAC6ā-actinin-2
eIF2ā TNNI2
PCNT
PIEZO1 Ă-Trcp2
ă-adaptin TPM1
PSMD7
PKD1L1
TRPP1 TRPP2 PKD1L3
FLNA IFT57
CD2AP
GJA1 NEK8 TRPP PHB2
DIAPH1
HAX1 ā-actinin-1 VCP TRPP3
TJP1
VASP WWTR1 CABP1 MDFI Gz-ā HERPUD1
āII-spectrin SLC25A4SLC8A1 DPYSL2 CALR ANXA1
EPB41 GRM1 CTTN PKHD1 RyR
ATP2A2 ITPR1Gāi2 JPH2 PLCă2 TRPM6
HOMER1
MARCKS DRD2 PKD1ā-actinin PLCĄ1
ORAI3 PLCĂ3DMD
FKBP5 RYR1 CAV3 ATP2B1
VAMP RNF24 WNK4 RHOA
GAP43
āSNAP
ATP2A3 ITPR2 TRPM7
ĂV-spectrin HOMER2
ITPR3 NYX
HOMER3 TRPC7 ăENaCRYR2 SLC2A4 TRPM
TRPC NCS1 ORAI2 RTP1
SLC9A3R1 TRPC4 UBC
STIM2 TRPC3 GNB2L1 CSNK1A1
SESTD1 EZR CDH5 MYH10
TRPC1 SNAP23 GPR179
CaMKIIĂ LYNTRPC5 SYPL2 MYH9MX1 TRPC2 PRKCSH PLCĂ1
PACS1 ABCC8
PRKG1 ASPH TRPM4
SHANK TARBP2 Gāq/11 FKBP1A SYN1 TRPM1
SLC9A3R2
Gāi3 CAV1
ă-actin
DYNC1H1 CASP4
VAMP2 CDH1
ā2-integrin
CACNA1C CASR STIM1
SRC SYT1 P2RX3 14-3-3ă
ENKUR DBN1ā1-syntrophin
STMN1 SNAP25
EPOR MAP7
NPR1 STX3SNAPIN
TRPC6 ITCH
CLTC Homer PDE3A HCK
AGTR1
CASP1 TRPV4PPP3CA FYN KCNMA1EGFR
STMN4
STMN2
FKBP4 ORAI1 LCK
NKAā1 CALM1 Ă-catenin Ă-arrestin
AQP2 PKCĂ TRPM3
NTRK2 Ă-tubulin-5
STMN3 SNF8 SYT4 ā-tubulin-1A
DNM1 PI3K-p85Ă
FGFR1 PPIB S100A10 TTRRPPAA11 AQP5 Tubulin
PRKACA YES1 OS9
NPHS2
ANO1
CHRM1 CaMKII
NPHS1 PTPN1 KCNQ2
PACSIN1 PACSIN3 TRPV3
GRIN2B
RAB9A āENaCTRPV6 AQP4
TRPV5 PDZD3 TRPV1 ā-tubulin-1B
TRPML NIPSNAP1 PACSIN2 MAP3K7
HSPA8 CYLD CDK5
CALB1 PKCā Ă-tubulin-3RGS2 Akt
PDCD6 AKAP5
NME1 SCG3 NOS3TRPV2 PKCą PRKD1/PKCĀCDC42 STIP1
PTEN KIF13B
RAC2 BSPRY RAB11A CBL
KL RAB11FIP3 PKC
TRPML2 PKARIIā
KvĂ2
DNAJC14 PEA15 TRPML3
NUMB KCNQ3 AMPKā2 CNR1
LAPTM4B TRPM2
TRPV PPARā NTRK1
FAF1 PI3K-p85ā
YIF1B TPCN1 GABARAP
PXMP2 TRPML1 LGALS1 TGM2 GRIN1
RAC1
PIRT
TPCN2
LAPTM4A ACBD3Tmem185a STOML1 PTPN13
SYNE1
HSP90AA1 GABARAPL2
PIP5K1A SLC50A1PEX16 ERGIC3 (BAE) EFHC1
TRPM8
RHOG = human
TMEM163
LAPTM5 SEC31A
= mouse/rat OPRM1
HTR1B
Gāq
Figure 6.1: TRP channel PPI network extracted from the TRIP Database. The majority of the shown PPIs are with rodent
proteins (grey nodes), whereas only a minority depicts PPIs with human proteins (purple nodes). Node colors of TRP subfamilies
are indicated in the figure. Network was rendered with Cytoscape 3.8.0.29,30
143
Chapter 6. Appendix
Table 6.1: List of human TRP channel protein interactors deposited in the TRIP database.29,30
Note, that not all TRP channels are represented, as the most experiments did not use human
proteins for PPI investigations.
TRP channel Protein interactor Experiment
TRPC1 Calmodulin Fusion protein-pull down assay
TRPC1 Caveolin-1 Fusion protein-pull down assay
TRPC1 D2R Fusion protein-pull down assay
TRPC1 FKBP52/FKBP4 Co-immunoprecipitation
TRPC1 FKBP52/FKBP4 Fusion protein-pull down assay
TRPC1 I-MFA Fusion protein-pull down assay
TRPC1 NCS-1 Fusion protein-pull down assay
TRPC1 TRPP1 Fusion protein-pull down assay
TRPC3 Calmodulin Fusion protein-pull down assay
TRPC3 FKBP12 Co-immunoprecipitation
TRPC3 Homer-1 Fusion protein-pull down assay
TRPC3 IP3R3 Fusion protein-pull down assay
TRPC3 MxA Fusion protein-pull down assay
TRPC3 NCX1 Fusion protein-pull down assay
TRPC3 Orai1 Fusion protein-pull down assay
TRPC3 TRPC1 Fusion protein-pull down assay
TRPC3 TRPM4 Yeast two-hybrid
TRPC4 αII-spectrin Fusion protein-pull down assay
TRPC4 αII-spectrin Fusion protein-pull down assay
TRPC4 D2R Fusion protein-pull down assay
TRPC4 MxA Fusion protein-pull down assay
TRPC4 NHERF1 Fusion protein-pull down assay
TRPC5 D2R Fusion protein-pull down assay
TRPC5 Gαqi3 Fusion protein-pull down assay
TRPC5 MxA Fusion protein-pull down assay
TRPC5 NCS-1 Yeast two-hybrid
TRPC5 NCS-1 Fusion protein-pull down assay
TRPC6 MxA Fusion protein-pull down assay
TRPC7 MxA Fusion protein-pull down assay
144
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPM3 Calmodulin Fluorescence probe labeling
TRPM3 S100A10 Fluorescence probe labeling
TRPM4 TRPC3 Yeast two-hybrid
TRPM7 SNAPIN Fusion protein-pull down assay
TRPM8 Gαq Fusion protein-pull down assay
TRPML1 ALG-2/PDCD6 Fusion protein-pull down assay
TRPML1 HSC70 Fusion protein-pull down assay
TRPML1 HSP40/DNAJC14 Fusion protein-pull down assay
TRPML1 LAPTM4a Yeast two-hybrid
TRPML1 LAPTM4b Yeast two-hybrid
TRPML1 LAPTM5 Yeast two-hybrid
TRPML1 SEC31A Fusion protein-pull down assay
TRPP1 ID2 Yeast two-hybrid
TRPP1 KIF3A Yeast two-hybrid
TRPP1 KIF3A Fusion protein-pull down assay
TRPP1 KIF3B Fusion protein-pull down assay
TRPP1 PACS-1 Fusion protein-pull down assay
TRPP1 PACS-2 Fusion protein-pull down assay
TRPP1 TRPP1 Yeast two-hybrid
TRPP1 RyR2 Fusion protein-pull down assay
TRPP1 Tropomyosin 1 Fusion protein-pull down assay
TRPP1 Troponin I 3 Fusion protein-pull down assay
TRPP1 TRPC1 Fusion protein-pull down assay
TRPP2 α-actinin-1 Yeast two-hybrid
TRPP2 α-actinin-1 Yeast two-hybrid
TRPP2 α-actinin-2 Yeast two-hybrid
TRPP2 α-actinin-2 Yeast two-hybrid
TRPP2 Troponin I 1 Fusion protein-pull down assay
TRPP2 Troponin I 2 Fusion protein-pull down assay
TRPP2 Troponin I 3 Fusion protein-pull down assay
TRPP3 RACK1 Fusion protein-pull down assay
TRPV1 AKAP5/AKAP150 Fusion protein-pull down assay
145
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 Calmodulin Fusion protein-pull down assay
Table 6.2: List of known TRPV4 interactors deposited in the TRIP database.29,30
TRP channel Protein interactor Experiment
TRPV4 LYN Co-immunoprecipitation
TRPV4 LYN Co-immunofluorescence staining
TRPV4 LYN In vitro PTM assay
TRPV4 LYN Calcium measurement
TRPV4 SRC Co-immunoprecipitation
TRPV4 SRC In vitro PTM assay
TRPV4 SRC Calcium measurement
TRPV4 FYN Co-immunoprecipitation
TRPV4 FYN Co-immunoprecipitation
TRPV4 FYN In vitro PTM assay
TRPV4 FYN Calcium measurement
TRPV4 HCK Co-immunoprecipitation
TRPV4 HCK In vitro PTM assay
TRPV4 HCK Calcium measurement
TRPV4 LCK Co-immunoprecipitation
TRPV4 LCK In vitro PTM assay
TRPV4 LCK Calcium measurement
TRPV4 YES Co-immunoprecipitation
TRPV4 YES In vitro PTM assay
TRPV4 YES Calcium measurement
TRPV4 LYN Inference
TRPV4 SRC Inference
TRPV4 FYN Inference
TRPV4 HCK Inference
TRPV4 LCK Inference
146
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 YES Inference
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 Calmodulin Patch clamp
TRPV4 Calmodulin Inference
TRPV4 MAP7 Yeast two-hybrid
TRPV4 MAP7 Co-immunoprecipitation
TRPV4 MAP7 Co-immunoprecipitation
TRPV4 MAP7 Co-immunofluorescence staining
TRPV4 MAP7 Co-immunofluorescence staining
TRPV4 MAP7 Co-immunofluorescence staining
TRPV4 MAP7 Cell surface biotinylation
TRPV4 MAP7 Patch clamp
TRPV4 PACS-1 Co-immunoprecipitation
TRPV4 PACS-1 Co-immunoprecipitation
TRPV4 PACS-1 Inference
TRPV4 NHERF4 Inference
TRPV4 AQP-5 Co-immunoprecipitation
TRPV4 AQP-5 Co-immunoprecipitation
TRPV4 AQP-5 Inference
TRPV4 Pacsin1 Yeast two-hybrid
TRPV4 Pacsin1 Co-immunoprecipitation
TRPV4 Pacsin1 Co-immunoprecipitation
TRPV4 Pacsin2 Inference
TRPV4 Pacsin2 Co-immunoprecipitation
TRPV4 Pacsin2 Co-immunoprecipitation
TRPV4 Pacsin3 Yeast two-hybrid
TRPV4 Pacsin3 Co-immunoprecipitation
TRPV4 Pacsin3 Co-immunoprecipitation
TRPV4 Pacsin3 Co-immunoprecipitation
TRPV4 Pacsin3 Co-immunoprecipitation
147
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 Pacsin3 Co-immunofluorescence staining
TRPV4 Pacsin3 Co-immunofluorescence staining
TRPV4 Pacsin3 Co-immunofluorescence staining
TRPV4 Pacsin3 Co-immunofluorescence staining
TRPV4 ITCH/AIP4 Co-immunoprecipitation
TRPV4 ITCH/AIP4 Co-immunofluorescence staining
TRPV4 ITCH/AIP4 Co-immunofluorescence staining
TRPV4 UBC In vivo PTM assay
TRPV4 ITCH/AIP4 Inference
TRPV4 UBC Inference
TRPV4 UBC In vivo PTM assay
TRPV4 OS-9 Yeast two-hybrid
TRPV4 OS-9 Co-immunoprecipitation
TRPV4 OS-9 Co-immunoprecipitation
TRPV4 OS-9 Co-immunofluorescence staining
TRPV4 OS-9 Cell surface biotinylation
TRPV4 Pacsin3 Co-immunoprecipitation
TRPV4 Pacsin2 Co-immunofluorescence staining
TRPV4 Pacsin3 Co-immunofluorescence staining
TRPV4 Pacsin3 Patch clamp
TRPV4 LYN Co-immunoprecipitation
TRPV4 α2-integrin Co-immunoprecipitation
TRPV4 α2-integrin Inference
TRPV4 IP3R3 Calcium measurement
TRPV4 IP3R3 Patch clamp
TRPV4 IP3R3 Co-immunoprecipitation
TRPV4 IP3R3 Inference
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunofluorescence staining
TRPV4 TRPP1 Fluorescence resonance energy transfer
TRPV4 TRPP1 Inference
TRPV4 TRPP1 Patch clamp
148
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 AKAP5/AKAP150 Co-immunoprecipitation
TRPV4 AKAP5/AKAP150 Speculation
TRPV4 IP3R3 Co-immunoprecipitation
TRPV4 IP3R3 Co-immunoprecipitation
TRPV4 IP3R3 Calcium measurement
TRPV4 IP3R3 Patch clamp
TRPV4 α-actin Fluorescence resonance energy transfer
TRPV4 α-actin Co-immunofluorescence staining
TRPV4 α-actin Inference
TRPV4 SRC In vivo PTM assay
TRPV4 SRC In vivo PTM assay
TRPV4 SRC Calcium measurement
TRPV4 80K-H Co-immunoprecipitation
TRPV4 80K-H Speculation
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 α-arrestin 2 Affinity purification-mass spectrometry
TRPV4 α-arrestin 1 Co-immunoprecipitation
TRPV4 α-arrestin 1 Inference
TRPV4 α-arrestin 1 Co-immunoprecipitation
TRPV4 AT1aR Inference
TRPV4 AT1aR Co-immunoprecipitation
TRPV4 AT1aR Co-immunofluorescence staining
TRPV4 AT1aR Co-immunoprecipitation
TRPV4 ITCH/AIP4 Co-immunoprecipitation
TRPV4 α-catenin Co-immunoprecipitation
TRPV4 E-cadherin Co-immunoprecipitation
TRPV4 α-catenin Yeast two-hybrid
TRPV4 E-cadherin Inference
TRPV4 α-catenin Co-immunoprecipitation
TRPV4 α-catenin Co-immunoprecipitation
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 Caveolin-1 Co-immunofluorescence staining
149
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 TRPC1 Inference
TRPV4 Caveolin-1 Inference
TRPV4 TRPC1 Co-immunofluorescence staining
TRPV4 TRPC1 Fluorescence resonance energy transfer
TRPV4 TRPC1 Co-immunoprecipitation
TRPV4 TRPC1 Calcium measurement
TRPV4 TRPC1 Co-immunoprecipitation
TRPV4 TRPC1 Co-immunofluorescence staining
TRPV4 TRPC1 Co-immunofluorescence staining
TRPV4 TRPC1 Calcium measurement
TRPV4 TRPC1 Cell surface biotinylation
TRPV4 TRPC1 Fluorescence resonance energy transfer
TRPV4 AQP-4 Inference
TRPV4 AQP-4 Co-immunofluorescence staining
TRPV4 AQP-4 Co-immunoprecipitation
TRPV4 AQP-4 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPP1 Atomic force microscopy(AFM) analysis
TRPV4 TRPC1 Fluorescence resonance energy transfer
TRPV4 TRPC1 Fluorescence resonance energy transfer
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 α-actin Co-immunoprecipitation
TRPV4 α-actin Fusion protein-pull down assay
TRPV4 Tubulin Co-immunoprecipitation
TRPV4 Tubulin Fusion protein-pull down assay
TRPV4 α-actin Inference
TRPV4 α-actin Fusion protein-pull down assay
TRPV4 Tubulin Fusion protein-pull down assay
TRPV4 α-tubulin-5 Inference
TRPV4 α-tubulin-5 Co-immunoprecipitation
TRPV4 α-tubulin-5 Co-immunofluorescence staining
TRPV4 E-cadherin Co-immunoprecipitation
150
Chapter 6. Appendix
TRP channel Protein interactor Experiment
TRPV4 α-catenin Co-immunoprecipitation
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunofluorescence staining
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunofluorescence staining
TRPV4 TRPP1 Calcium measurement
TRPV4 RPS27A Affinity purification-mass spectrometry
TRPV4 Myosin-HIIA Affinity purification-mass spectrometry
TRPV4 Myosin-10 Affinity purification-mass spectrometry
TRPV4 Calmodulin Fusion protein-pull down assay
TRPV4 Myosin-HIIA Co-immunofluorescence staining
TRPV4 Myosin-10 Co-immunofluorescence staining
TRPV4 TRPP1 Co-immunofluorescence staining
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 Pacsin3 Fluorescence resonance energy transfer
TRPV4 TMEM16A/ANO1 Inference
TRPV4 TMEM16A/ANO1 Co-immunoprecipitation
TRPV4 AKAP5/AKAP150 Co-immunofluorescence staining
TRPV4 AKAP5/AKAP150 Patch clamp
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPC1 Co-immunoprecipitation
TRPV4 TRPP1 Co-immunoprecipitation
TRPV4 TRPC1 Co-immunoprecipitation
TRPV4 TRPP1 Fluorescence resonance energy transfer
TRPV4 TRPC1 Fluorescence resonance energy transfer
TRPV4 TRPP1 Patch clamp
TRPV4 TRPC1 Patch clamp
TRPV4 Bkca Inference
TRPV4 Bkca Co-immunoprecipitation
TRPV4 TRPC1 Co-immunoprecipitation
151
Chapter 6. Appendix
6.2 Appendix - Purification of biotinylated TRPV4 N-terminal
proteins
150 hsV4 ARD wt Avi
hsV4 ARD R232C Avi
hsV4 ARD K276E Avi
M
[kDa] Coomassie
100
50
37
25
50 wt
2C 6E
23
R 2
7
K
Superdex 75
0
40 50 60 70 80 90 100
Volume [mL]
Figure 6.2: Purification of unbiotinylated hsV4 ARD Avi constructs. A SEC purifications of unbiotinylated hsV4 ARD Avi
constructs. For biotinylated constructs see figure 4.1. SEC runs were either performed with a HiLoad Superdex 75 pg preparative
SEC column. Inlet shows Coomassie stained 15 % SDS-PAGE of collected and concentrated fractions after SEC. x-axis: eluted
volume in mL, y-axis: absorbency at 280 nm, M = marker.
6.3 Appendix - A beginning to elucidate the versatile TRPV4
interatcome
Following tables contain the mass spec data of all significant identified protein interactome
members of respective TRPV4 N-terminal construct. With gene ontology and manual cura-
tion, each protein was investigated upon a possible role in RNP and/or stress granules. These
tables present the current data situation in April 2021 and do not claim any comprehensive-
ness.
152
Abs [mAu]
280nm
Chapter 6. Appendix
153
Table 6.3: List of cytoplasmic RNP granule and stress granule proteins found in the hsV4N wt Avi protein interactome determined via two
independent UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of each of
the two UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4N Avi wt (control) of each UDMSE
measurement. Bold numbers assign p- and log2 FC values to respective UDMSE measurement. Manual data curation was performed to
determine association of detected proteins with cytosolic ribonucleoprotein (RNP) and/or stress granules (see also figure 4.4). Respective
literature is appended (Lit.).
UniProt ID -log10 (p-value) 1 log2 FC 1 -log10 (p-value) 2 log2 FC 2 RNP granules Stress granules Lit.
TRPV4_HUMAN 6.69 5.59 8.81 4.95 no no
ABCD3_HUMAN 2.49 16.10 8.15 11.86 no no
ATD3B_HUMAN 6.31 19.30 1.38 11.81 no no
COPA_HUMAN 4.36 15.38 1.32 15.73 no no
COX5B_HUMAN 4.25 17.66 5.43 8.72 no no
DDX3X_HUMAN 4.83 17.06 2.38 13.00 yes yes 107–109,120,127,200–203
DDX5_HUMAN 2.77 17.53 5.94 14.63 no yes 107,203,204
DHX9_HUMAN 1.49 15.05 7.22 14.77 yes yes 108,120
HNRH1_HUMAN 7.02 18.54 6.60 11.31 no yes 108,200,205
HNRPF_HUMAN 4.35 18.24 6.25 11.34 no yes 108,205
HNRPM_HUMAN 3.03 18.57 6.27 4.44 no no
HNRPU_HUMAN 5.91 18.05 1.75 13.27 yes yes 108,200,206
IF2B1_HUMAN 2.84 15.38 9.89 12.93 yes yes 107,108,120,200
PABP1_HUMAN 4.21 16.89 5.45 14.21 yes yes 107–109,207–209
PACN3_HUMAN 5.85 15.59 4.10 11.90 no no
PHB2_HUMAN 5.20 4.12 4.10 5.54 no no
PLEC_HUMAN 2.60 18.36 4.23 14.25 no no
PRKDC_HUMAN 1.34 18.58 5.88 16.38 no no
Chapter 6. Appendix
154
UniProt ID -log10 (p-value) 1 log2 FC 1 -log10 (p-value) 2 log2 FC 2 RNP granules Stress granules Lit
RBM14_HUMAN 3.53 17.89 2.74 12.17 no no
RL13_HUMAN 2.69 18.92 5.41 14.44 no no
RL17_HUMAN 1.56 18.81 5.58 13.53 no no
RL24_HUMAN 1.96 18.14 3.18 12.19 no no
RL28_HUMAN 6.63 17.41 1.97 13.40 yes no 210
RL35_HUMAN 2.41 16.58 3.39 11.41 no no
RL36_HUMAN 1.44 15.75 7.46 11.89 no no
ROA2_HUMAN 5.33 17.21 5.69 12.44 yes yes 107,108,126,200,206,211
RS2_HUMAN 2.57 2.05 4.31 4.89 no no
RS27A_HUMAN 2.56 18.47 6.68 11.46 no no
RS6_HUMAN 2.62 18.19 5.73 5.14 yes yes 113,210,212
RS7_HUMAN 1.81 15.64 5.36 11.97 no no
RS8_HUMAN 2.61 18.47 2.53 4.66 no no
RT17_HUMAN 6.47 14.91 2.24 10.84 no no
SYNE1_HUMAN 1.74 18.41 7.64 4.40 yes no 210
VDAC2_HUMAN 7.64 19.32 3.04 10.64 no no
YBOX1_HUMAN 4.72 17.37 8.27 13.50 yes yes 107,108,116,120,206,213
Chapter 6. Appendix
155
Table 6.4: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4N protein interactome in the presence of 2 mM
Ca2+ determined via UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of
each of the two UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4N wt Avi (control) of each
UDMSE measurement. Bold numbers assign p- and log2 FC values to respective UDMSE measurement. Manual data curation was performed
to determine association of detected proteins with cytosolic ribonucleoprotein (RNP) and/or stress granules (see also figure 4.5)
UniProt ID -log10 (p-value) 1 log2 FC 1 -log10 (p-value) 2 log2 FC 2 RNP granules Stress granules Lit.
TRPV4_HUMAN 4.09 5.22 7.84 4.84 no no
ABCD3_HUMAN 2.83 15.59 7.01 12.56 no no
ADT2_HUMAN 4.12 2.23 5.92 14.68 no no
ATD3A_HUMAN 2.92 3.57 4.01 14.81 no yes 107
DDX3X_HUMAN 4.12 16.08 4.74 12.83 no yes 107–109,120,127,200–203
DDX5_HUMAN 11.47 16.85 3.87 15.03 no yes 107,203,204
DHX9_HUMAN 6.87 14.80 5.64 15.59 yes yes 108,120
EPIPL_HUMAN 5.86 19.97 5.59 16.62 no no
HNRH1_HUMAN 5.38 18.46 9.19 11.61 no yes 108,200
HNRPF_HUMAN 3.10 17.89 6.14 11.59 no yes 108
HNRPM_HUMAN 4.38 17.72 4.75 4.66 no no
HNRPU_HUMAN 7.60 17.83 2.39 2.35 yes yes 108,200,206
IF2B1_HUMAN 7.88 14.88 2.50 12.83 yes yes 107,108,120,200
KHDR1_HUMAN 6.30 16.40 3.18 11.28 no yes 107,124
KPYM_HUMAN 1.92 2.38 2.32 1.65 no no
NFM_HUMAN 10.16 5.63 1.89 3.25 no no
NPM_HUMAN 3.49 17.29 3.24 2.55 no no
PABP1_HUMAN 2.93 16.51 4.24 14.47 yes yes 107–109,207–209
PACN3_HUMAN 3.80 14.89 3.61 11.80 no no
PHB_HUMAN 5.52 19.99 6.46 12.40 no no
Chapter 6. Appendix
156
UniProt ID -log10 (p-value) 1 log2 FC 1 -log10 (p-value) 2 log2 FC 2 RNP granules Stress granules Lit
PHB2_HUMAN 4.42 3.91 6.45 6.23 no no
PLEC_HUMAN 4.01 17.17 3.89 14.02 no no
PRKDC_HUMAN 2.43 18.18 4.82 17.27 no no
RBM14_HUMAN 5.47 16.65 1.54 12.23 no no
RBM8A_HUMAN 2.98 15.49 6.78 12.36 no no
RL10_HUMAN 6.07 18.16 7.41 14.58 no no
RL11_HUMAN 3.94 2.38 6.05 12.75 no yes 108
RL12_HUMAN 7.93 17.25 7.36 12.75 no no
RL13A_HUMAN 4.63 2.11 6.83 12.87 no no
RL15_HUMAN 4.99 17.13 3.12 13.58 no no
RL17_HUMAN 7.40 17.62 6.28 12.85 no no
RL18_HUMAN 3.86 17.85 4.01 13.18 no no
RL18A_HUMAN 3.59 1.98 7.71 14.77 no no
RL21_HUMAN 1.41 16.53 6.67 13.64 no no
RL24_HUMAN 5.33 18.21 7.50 11.93 no no
RL27_HUMAN 2.48 16.29 2.96 11.82 no no
RL28_HUMAN 5.31 16.46 5.06 13.23 yes no 210
RL3_HUMAN 3.59 1.35 2.75 3.50 no no
RL30_HUMAN 1.63 16.97 2.37 4.89 no no
RL32_HUMAN 6.36 16.23 6.67 12.51 no no
RL34_HUMAN 3.70 3.59 1.87 12.69 no no
RL35_HUMAN 3.99 16.07 5.67 8.49 no no
RL36_HUMAN 4.11 15.61 7.19 11.17 no no
RL36A_HUMAN 2.19 1.22 5.42 12.08 no no
RL37A_HUMAN 8.27 17.13 8.85 10.63 no no
RL5_HUMAN 1.61 2.81 8.25 14.26 no no
Chapter 6. Appendix
157
UniProt ID -log10 (p-value) 1 log2 FC 1 -log10 (p-value) 2 log2 FC 2 RNP granules Stress granules Lit
RL6_HUMAN 3.60 3.18 6.88 14.93 yes no 210
RL7A_HUMAN 5.69 16.96 3.85 13.91 no no
RLA0_HUMAN 4.79 16.54 6.53 12.11 yes no 206,214
ROA2_HUMAN 8.18 17.08 2.05 2.29 yes yes 107,108,126,200,206,211
RS11_HUMAN 4.41 19.00 5.67 10.46 no no
RS27A_HUMAN 5.65 17.35 8.03 11.64 no no
RS27L_HUMAN 4.80 15.97 2.52 3.21 no no
RS4X_HUMAN 6.93 17.79 8.12 13.23 yes no 206
RS6_HUMAN 5.88 17.81 2.69 5.23 yes yes 113,210,212
RS8_HUMAN 9.09 18.15 4.79 4.53 no no
RS9_HUMAN 2.51 17.21 4.21 12.63 no no
RT17_HUMAN 1.72 14.63 3.27 11.15 no no
SYNE1_HUMAN 2.91 18.11 2.85 3.09 yes no 210
TBA3E_HUMAN 4.46 19.93 8.46 14.18 no no
TBA4A_HUMAN 4.55 17.05 2.41 3.24 no yes 107
TBB4B_HUMAN 5.07 20.09 3.35 4.00 no no
VDAC2_HUMAN 5.26 17.83 6.93 11.54 no no
VIME_HUMAN 6.02 3.60 3.37 5.48 no no
YBOX1_HUMAN 12.38 17.03 1.50 12.50 yes yes 107,108,116,120,206,213
Chapter 6. Appendix
158
Table 6.5: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD wt protein interactome determined via
UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of each of the two
UDMSE measurements, log2 fold change (log E2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi (control) of each UDMS
measurement. Manual data curation was performed to determine association of detected proteins with cytosolic ribonucleoprotein (RNP)
and/or stress granules (see also figure 4.6 A)
UniProt ID -log10 (p-value) 1 log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 2,13 15,77 no no
1433E_HUMAN 8,32 13,01 no no
1433T_HUMAN 12,43 13,15 no no
ACLY_HUMAN 3,74 1,30 no no
AK1A1_HUMAN 17,63 11,13 no no
BOLA2_HUMAN 7,94 12,21 no no
CAH2_HUMAN 3,41 2,46 no no
COPD_HUMAN 6,28 12,09 no no
DDX3X_HUMAN 2,13 0,98 no yes 107–109,120,127,200–203
DNJA1_HUMAN 2,74 1,24 no yes 107
ENOG_HUMAN 6,00 2,03 no no
ERF3A_HUMAN 3,28 11,60 no no
GSTP1_HUMAN 2,38 2,36 no no
H31T_HUMAN 1,33 1,24 no no
HNRH2_HUMAN 10,58 12,40 no yes 107
HNRPC_HUMAN 2,03 1,35 no no
IF2B2_HUMAN 10,69 12,87 yes yes 107–109,120,204,215
IF4A1_HUMAN 3,16 1,19 no yes 107,108,216
MDHC_HUMAN 4,61 1,20 no no
NONO_HUMAN 3,62 1,28 yes yes 107,217
Chapter 6. Appendix
159
UniProt ID -log10 (p-value) 1 log2 FC Lit.
NPM_HUMAN 1,93 1,14 no no
PEBP1_HUMAN 9,26 11,37 no no
POTEE_HUMAN 8,02 12,79 no no
POTEJ_HUMAN 7,70 10,43 no no
PRDX6_HUMAN 6,73 11,79 no yes 107
PUR6_HUMAN 2,17 1,54 no no
PUR9_HUMAN 9,44 11,06 no no
RL11_HUMAN 1,92 1,39 no no
RL27A_HUMAN 1,37 1,03 no no
RL4_HUMAN 1,77 1,22 no no
RL5_HUMAN 1,45 1,48 no no
ROA2_HUMAN 1,65 0,97 no yes 107,108,126,211
RS13_HUMAN 1,86 1,36 no no
SRSF3_HUMAN 1,60 1,02 no yes 211
SRSF8_HUMAN 4,03 1,29 no no
TBA4A_HUMAN 4,19 1,04 no yes 107,129,130
TBB4A_HUMAN 6,67 11,30 no no
TCPD_HUMAN 5,09 1,61 no no
TIAR_HUMAN 7,76 11,87 no yes 107–109,205,207,218–220
UBP2L_HUMAN 1,36 1,38 no yes 107–109,221
YTHD3_HUMAN 2,49 1,28 no yes 107–109,222
Chapter 6. Appendix
160
Table 6.6: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD wt protein interactome in the presence of 2 mM
Ca2+ determined via UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of
each of the two UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi (control) of each
UDMSE measurement. Manual data curation was performed to determine association of detected proteins with cytosolic ribonucleoprotein
(RNP) and/or stress granules (see also figure 4.6 B).
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 7,59 15,46 no no
1433E_HUMAN 22,08 12,87 no no
1433G_HUMAN 4,82 2,04 no no
1433T_HUMAN 13,78 11,98 no no
ACLY_HUMAN 2,68 1,82 no no
AK1A1_HUMAN 6,24 11,36 no no
ALDOA_HUMAN 2,14 1,39 no no
BOLA2_HUMAN 6,14 13,05 no no
CAH2_HUMAN 4,08 2,50 no no
COF1_HUMAN 1,69 1,19 no no
COF2_HUMAN 2,19 2,08 no no
COPD_HUMAN 5,54 12,27 no no
DDX1_HUMAN 1,38 0,96 no yes 107–109,116
DDX3X_HUMAN 2,36 1,09 no yes 107–109,120,127,200–203
DDX5_HUMAN 2,14 1,22 yes yes 203,204
DNJA1_HUMAN 2,74 1,24 no yes 107
DNJC7_HUMAN 2,82 1,45 no no
DUT_HUMAN 3,92 1,75 no no
ENOG_HUMAN 2,27 2,80 no no
ENPL_HUMAN 2,04 1,02 no no
Chapter 6. Appendix
161
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
ERF3A_HUMAN 8,27 11,95 no no
FKB1A_HUMAN 2,36 2,19 no no
G3BP1_HUMAN 3,06 1,93 no yes 107–109,123,126,135,204,205,211,223
G3BP2_HUMAN 1,52 1,22 no yes 223,224
GSTP1_HUMAN 2,90 2,32 no no
H31T_HUMAN 1,64 0,97 no no
HNRH2_HUMAN 5,55 12,90 no yes 107
HNRPF_HUMAN 1,63 1,13 no yes 108
HNRPM_HUMAN 1,54 1,08 no yes 108
HS90A_HUMAN 1,04 1,50 no no
HSP76_HUMAN 3,27 1,28 no no
IF2B_HUMAN 2,66 1,85 no yes 107
IF2B2_HUMAN 11,80 14,12 yes yes 107–109,120,204,215
IF4A1_HUMAN 1,74 3,65 no yes 107,108,205,216
IF4G2_HUMAN 1,86 0,96 no yes 107,109
IF5A2_HUMAN 1,54 1,14 no yes 225
KCD12_HUMAN 2,71 1,31 no no
LC7L2_HUMAN 1,13 1,00 no no
LDHA_HUMAN 2,77 1,60 no no
LDHB_HUMAN 2,26 1,48 no no
MDHC_HUMAN 3,07 1,89 no no
NONO_HUMAN 2,67 1,76 yes yes 107,217
NPM_HUMAN 3,15 1,62 no no
NUCL_HUMAN 1,73 1,15 no no
PABP1_HUMAN 1,92 1,15 no yes 107–109,127,202,226
PABP4_HUMAN 1,30 1,01 no no
Chapter 6. Appendix
162
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
PDIA3_HUMAN 1,67 1,75 no yes 108
PEBP1_HUMAN 10,73 11,94 no no
POTEE_HUMAN 13,55 13,94 no no
POTEJ_HUMAN 11,06 11,50 no no
PRDX1_HUMAN 2,66 1,21 no yes 107,108
PRDX2_HUMAN 11,42 13,82 no no
PRDX6_HUMAN 5,10 12,41 no yes 107
PTBP1_HUMAN 2,05 1,45 no no
PUR6_HUMAN 3,35 2,40 no no
PUR9_HUMAN 15,84 11,19 no no
RAN_HUMAN 1,69 1,22 no no
RBM39_HUMAN 0,91 0,83 no no
RL11_HUMAN 4,04 1,58 no no
RL12_HUMAN 2,51 1,19 no no
RL13_HUMAN 2,62 1,12 no no
RL15_HUMAN 2,66 1,24 no no
RL18A_HUMAN 2,61 1,31 no no
RL21_HUMAN 3,30 1,38 no no
RL23A_HUMAN 3,20 1,24 no no
RL27A_HUMAN 1,65 0,95 no no
RL28_HUMAN 2,12 1,49 no no
RL3_HUMAN 4,54 1,62 no no
RL34_HUMAN 1,61 1,22 no no
RL36L_HUMAN 4,02 2,06 no no
RL37A_HUMAN 3,38 1,37 no no
RL4_HUMAN 5,13 1,61 no no
Chapter 6. Appendix
163
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
RL5_HUMAN 4,83 1,88 no no
RL6_HUMAN 2,00 1,10 no no
RL7A_HUMAN 2,42 1,29 no no
RLA0L_HUMAN 3,55 1,54 no no
ROA1_HUMAN 2,99 1,10 no yes 107,108,219
RS11_HUMAN 3,84 1,45 no no
RS13_HUMAN 3,88 1,63 no no
RS15A_HUMAN 7,06 2,12 no no
RS20_HUMAN 1,67 1,24 no no
RS21_HUMAN 1,98 1,02 no no
RS23_HUMAN 1,48 1,38 no no
RS28_HUMAN 1,54 1,10 no no
RS30_HUMAN 3,49 1,19 no no
RS5_HUMAN 4,20 1,35 no no
RS6_HUMAN 1,24 0,91 no no
SRSF3_HUMAN 1,84 0,87 no yes 211
SRSF8_HUMAN 4,38 1,70 no no
TAGL2_HUMAN 2,92 1,25 no no
TBB3_HUMAN 2,23 1,92 no yes 107
TBB4A_HUMAN 5,94 10,47 no no
TBB6_HUMAN 1,73 1,49 no no
TCPA_HUMAN 2,21 1,72 no no
TCPB_HUMAN 2,17 1,37 no no
TCPD_HUMAN 3,32 1,75 no no
TCPG_HUMAN 3,53 1,82 no no
TCPH_HUMAN 1,94 1,29 no no
Chapter 6. Appendix
164
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
TCPZ_HUMAN 4,15 1,61 no no
TEBP_HUMAN 4,49 1,80 no no
TIAR_HUMAN 6,73 12,86 no yes 107–109,205,207,218–220
TPIS_HUMAN 2,68 1,71 no no
U2AF2_HUMAN 5,17 13,73 no yes 107
UBP2L_HUMAN 5,60 1,71 no yes 107–109,205,221
Chapter 6. Appendix
165
Table 6.7: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD R232C protein interactome determined via
UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of each of the two
UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi (control) of each UDMSE
measurement. Manual data curation was performed to determine association of detected proteins with cytosolic ribonucleoprotein (RNP)
and/or stress granules (see also figure 4.6 C).
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 11,53 17,45 no no
1433F_HUMAN 1,33 1,18 no no
DDX3X_HUMAN 5,06 2,18 no yes 107–109,120,127,200–203
IF4A3_HUMAN 10,43 11,91 no yes 108
NONO_HUMAN 2,01 2,05 yes yes 107,217
POTEJ_HUMAN 1,42 5,94 no no
RL11_HUMAN 3,27 1,38 no no
RL3_HUMAN 1,74 1,49 no no
RL35A_HUMAN 2,93 1,51 no no
RLA0_HUMAN 1,37 1,01 no no
RS15A_HUMAN 2,22 1,12 no no
RS6_HUMAN 1,48 1,05 no no
SFPQ_HUMAN 3,65 1,92 no yes 107,217
SRSF7_HUMAN 1,49 1,38 no yes 227
U2AF2_HUMAN 2,55 1,58 no yes 107
Chapter 6. Appendix
166
Table 6.8: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD R232C protein interactome in the presence
of 2 mM Ca2+ determined via UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadru-
plicates of each of the two UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi
(control) of each UDMSE measurement. Manual data curation was performed to determine association of detected proteins with cytosolic
ribonucleoprotein (RNP) and/or stress granules (see also figure 4.6 C).
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 4,89 18,44 no no
1433F_HUMAN 3,02 1,98 no no
DDX17_HUMAN 3,17 1,73 no yes 204
DDX3X_HUMAN 6,12 2,94 no yes 107–109,120,127,200–203
IF2B_HUMAN 2,19 1,49 no yes 107
IF4A3_HUMAN 2,94 12,58 no yes 108
KCD12_HUMAN 1,63 1,47 no no
NONO_HUMAN 4,48 2,45 yes yes 107,217
POTEE_HUMAN 3,80 4,40 no no
POTEJ_HUMAN 4,74 6,57 no no
RL11_HUMAN 3,36 2,19 no no
RL12_HUMAN 2,41 1,56 no no
RL13_HUMAN 1,34 1,64 no no
RL15_HUMAN 4,91 1,99 no no
RL18A_HUMAN 1,63 2,13 no no
RL24_HUMAN 1,72 1,83 no no
RL29_HUMAN 4,07 1,83 no no
RL3_HUMAN 3,57 2,42 no no
RL34_HUMAN 3,07 2,04 no no
RL37A_HUMAN 5,22 2,03 no no
Chapter 6. Appendix
167
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
RL4_HUMAN 2,02 2,38 no no
RL5_HUMAN 2,31 2,34 no no
RL6_HUMAN 1,64 1,91
RL7A_HUMAN 1,62 1,95 no no
ROA1_HUMAN 2,89 1,90 no yes 107,108,219
ROA2_HUMAN 1,38 1,43 no yes 107,108,126,211
RS13_HUMAN 2,99 1,75 no no
RS15A_HUMAN 4,13 1,79 no no
RS2_HUMAN 1,31 1,34 no no
RS20_HUMAN 2,06 1,56 no no
RS26L_HUMAN 1,71 1,96 no no
RS6_HUMAN 1,52 2,17 no no
RUVB1_HUMAN 2,12 1,62 no no
SFPQ_HUMAN 4,23 2,37 no yes 107,217
SRSF2_HUMAN 2,44 2,68 no no
SRSF3_HUMAN 2,80 1,66 no yes 211
SRSF7_HUMAN 4,13 3,02 no yes 227
TCPG_HUMAN 1,81 1,48 no no
Chapter 6. Appendix
168
Table 6.9: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD K276E protein interactome determined via
UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadruplicates of each of the two
UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi (control) of each UDMSE
measurement. Manual data curation was performed to determine association of detected proteins with cytosolic ribonucleoprotein (RNP)
and/or stress granules (see also figure 4.6 C).
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 3,53 7,08 no no
1433B_HUMAN 5,12 12,38 no no
1433G_HUMAN 1,41 1,66 no no
ACTBL_HUMAN 7,69 12,25 no no
ACTS_HUMAN 5,49 13,82 no no
AK1A1_HUMAN 3,91 12,76 no no
ANXA5_HUMAN 6,41 12,13 no no
BOLA2_HUMAN 1,56 1,10 no no
CAPR1_HUMAN 4,12 1,70 no no
DDX1_HUMAN 1,73 0,98 no yes 107–109,116
DDX17_HUMAN 3,21 1,37 no yes 204
DDX3X_HUMAN 7,16 2,41 no yes 107–109,120,127,200–203
DDX5_HUMAN 2,55 1,09 yes yes 203,204
DNJA1_HUMAN 2,86 0,93 no yes 107
ENSA_HUMAN 3,33 13,13 no no
FKB1A_HUMAN 1,74 1,41 no no
G3BP1_HUMAN 1,62 1,31 no yes 107–109,123,126,135,204,205,211,223
G3BP2_HUMAN 3,10 2,41 no yes 223,224
GGYF2_HUMAN 2,53 1,26 no no
HAP28_HUMAN 2,79 1,76 no no
Chapter 6. Appendix
169
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
HNRPU_HUMAN 1,94 1,23 yes yes 108,200,206
HS71A_HUMAN 0,06 1,10 no no
HS74L_HUMAN 3,66 11,83 no no
HS904_HUMAN 6,04 12,64 no no
HSP72_HUMAN 4,49 2,04 no no
IF2B2_HUMAN 10,10 13,62 yes yes 107–109,120,204,215
IF4A2_HUMAN 7,57 15,63 no yes 228
IF4A3_HUMAN 3,23 3,69 no yes 108
JUPI2_HUMAN 1,86 0,68 no no
KHDR1_HUMAN 5,59 2,13 no yes 107,124
KR111_HUMAN 4,76 14,13 no no
LKHA4_HUMAN 5,22 1,72 no no
MDHC_HUMAN 1,90 1,38 no no
MDHM_HUMAN 9,21 12,11 no no
METK2_HUMAN 2,32 1,03 no no
MOES_HUMAN 1,35 1,16 no no
NOLC1_HUMAN 3,00 0,90 no yes 108
NONO_HUMAN 5,42 1,97 yes yes 107,217
NUCL_HUMAN 1,36 1,58 no no
NUDC_HUMAN 3,48 0,01 no yes 107
PAL4A_HUMAN 1,69 2,65 no no
PAP1L_HUMAN 1,51 0,95 no no
PEBP1_HUMAN 4,28 2,25 no no
POTEF_HUMAN 1,79 1,63 no no
PRDX4_HUMAN 2,55 1,23 no no
PRDX6_HUMAN 4,31 12,78 no yes 107
Chapter 6. Appendix
170
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
PUR9_HUMAN 5,26 2,02 no no
RBM14_HUMAN 1,28 0,92 no no
RBM39_HUMAN 1,34 0,75 no no
RBM4_HUMAN 2,29 1,07 no no
RL10A_HUMAN 2,30 1,95 no no
RL11_HUMAN 5,22 1,88 no no
RL12_HUMAN 1,70 0,97 no no
RL13_HUMAN 2,79 1,56 no no
RL15_HUMAN 4,42 1,63 no no
RL18_HUMAN 1,49 2,37 no no
RL18A_HUMAN 2,60 1,81 no no
RL19_HUMAN 3,16 1,82 no no
RL24_HUMAN 3,06 1,74 no no
RL27_HUMAN 1,58 1,94 no no
RL28_HUMAN 3,24 2,15 no no
RL29_HUMAN 2,91 1,14 no no
RL3_HUMAN 4,03 2,08 no no
RL34_HUMAN 3,00 1,72 no no
RL35A_HUMAN 2,70 1,47 no no
RL36_HUMAN 2,28 1,62 no no
RL36A_HUMAN 3,13 1,81 no no
RL36L_HUMAN 4,07 1,76 no no
RL4_HUMAN 4,62 1,95 no no
RL5_HUMAN 3,94 1,72 no no
RL6_HUMAN 2,81 1,32 no no
RL7_HUMAN 3,39 13,95 no no
Chapter 6. Appendix
171
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
RL7A_HUMAN 3,26 1,45 no no
RL8_HUMAN 1,37 1,20 no no
RLA0L_HUMAN 2,37 1,92 no no
RMXL1_HUMAN 7,10 12,71 no no
ROA1_HUMAN 4,19 1,80 no yes 107,108,219
ROA2_HUMAN 2,09 1,44 no yes 107,108,219
RS11_HUMAN 2,05 1,51 no no
RS12_HUMAN 7,86 1,93 no no
RS13_HUMAN 3,94 1,48 no no
RS15A_HUMAN 3,35 1,29 no no
RS16_HUMAN 1,55 1,14 no no
RS17_HUMAN 2,09 1,44 no no
RS19_HUMAN 1,67 1,92 no no
RS20_HUMAN 2,13 1,45 no no
RS23_HUMAN 3,59 1,80 no no
RS24_HUMAN 1,47 1,40 no no
RS26L_HUMAN 2,26 1,43 no no
RS3_HUMAN 2,99 1,40 no no
RS30_HUMAN 3,81 1,70 no no
RS3A_HUMAN 2,19 1,48 no no
RS4X_HUMAN 1,39 0,84 no no
RS5_HUMAN 2,83 1,43 no no
RS6_HUMAN 2,50 2,01 no no
RS7_HUMAN 2,99 1,47 no no
RS8_HUMAN 2,25 1,47 no no
RS9_HUMAN 2,56 1,32 no no
Chapter 6. Appendix
172
UniProt ID -log10 (p-value) log2 FC RNP granule Stress granules Lit.
RUVB2_HUMAN 1,54 1,90 no no
SFPQ_HUMAN 3,76 1,61 no yes 107,217
SRP09_HUMAN 1,98 2,02 no no
SRSF1_HUMAN 3,40 1,95 no no
SRSF3_HUMAN 1,95 1,09 no yes 211
SRSF6_HUMAN 3,63 2,00 no no
SRSF7_HUMAN 4,73 2,40 no yes 227
STAU1_HUMAN 1,58 0,93 no yes 207
STK26_HUMAN 2,82 0,44 no no
SYNC_HUMAN 2,88 13,32 no no
TBB2B_HUMAN 5,59 2,16 no no
TBB6_HUMAN 1,82 1,11 no no
TERA_HUMAN 2,78 2,18 no no
TR150_HUMAN 3,15 2,90 no no
U2AF1_HUMAN 5,87 1,76 no yes 107
U2AF2_HUMAN 8,24 3,79 no no
Chapter 6. Appendix
173
Table 6.10: List of cytoplasmic hRNP granule and stress granule proteins found in the hsV4 ARD K276E protein interactome in the presence
of 2 mM Ca2+ determined via UDMSE in this work. p-values (Benjamini-Hochberg corrected Student’s t-test) were determined of the quadru-
plicates of each of the two UDMSE measurements, log2 fold change (log2 FC) between biotinylated and non-biotinylated hsV4 ARD wt Avi
(control) of each UDMSE measurement. Manual data curation was performed to determine association of detected proteins with cytosolic
ribonucleoprotein (RNP) and/or stress granules (see also figure 4.6 C).
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
TRPV4_HUMAN 12,31 6,54 no no
ACLY_HUMAN 2,08 0,85 no no
AK1A1_HUMAN 9,88 12,05 no no
CAH2_HUMAN 4,39 1,19 no no
DDX17_HUMAN 4,46 1,40 no yes 204
DDX3X_HUMAN 8,77 2,58 no yes 107–109,120,127,200–203
DDX5_HUMAN 3,01 1,19 yes yes 203,204
DNJA1_HUMAN 2,41 0,90 no yes 107
ENOG_HUMAN 3,68 1,70 no no
G3BP1_HUMAN 1,53 0,95 no yes 107–109,123,126,135,204,205,211,223
G3BP2_HUMAN 4,44 2,67 no yes 223,224
GSTP1_HUMAN 1,50 1,05 no no
HNRPU_HUMAN 2,70 0,92 yes yes 108,200,206
IF2B2_HUMAN 9,62 13,81 yes yes 107–109,120,204,215
IF4A2_HUMAN 11,20 14,24 no yes 228
IF4A3_HUMAN 6,41 2,94 no yes 108
IF4B_HUMAN 1,63 0,94 no yes 107,108,205
KCRB_HUMAN 3,11 1,32 no no
KHDR1_HUMAN 5,13 2,33 no yes 107,124
LARP1_HUMAN 1,57 0,94 no yes 107
Chapter 6. Appendix
174
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
LDHA_HUMAN 1,53 1,02 no no
NPM_HUMAN 1,20 1,13 no no
NUCL_HUMAN 2,76 1,09 no no
PABP4_HUMAN 1,49 0,84 no yes 107–109
PAL4A_HUMAN 2,95 2,17 no no
PAP1L_HUMAN 2,73 1,01 no no
PGK1_HUMAN 2,99 1,06 no no
POTEF_HUMAN 2,18 1,21 no no
PRDX6_HUMAN 7,42 12,63 no yes 107
PUR9_HUMAN 2,78 1,30 no no
RBM39_HUMAN 2,78 1,11 no no
RBM4_HUMAN 1,46 0,94 no no
RCC2_HUMAN 4,46 13,25 no no
RL10A_HUMAN 2,79 1,43 no no
RL11_HUMAN 5,27 1,89 no no
RL13_HUMAN 4,57 1,46 no no
RL15_HUMAN 4,64 1,47 no no
RL18_HUMAN 4,07 1,64 no no
RL18A_HUMAN 4,58 1,59 no no
RL19_HUMAN 5,36 1,68 no no
RL23A_HUMAN 1,02 0,75 no no
RL27_HUMAN 5,24 1,67 no no
RL28_HUMAN 5,55 2,00 no no
RL3_HUMAN 6,66 2,00 no no
RL35A_HUMAN 4,43 1,42 no no
RL36_HUMAN 4,32 1,63 no no
Chapter 6. Appendix
175
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
RL4_HUMAN 5,76 1,80 no no
RL5_HUMAN 4,90 1,75 no no
RL6_HUMAN 2,80 1,15 no no
RL7_HUMAN 8,39 13,76 no no
RLA0L_HUMAN 6,38 1,68 no no
RMXL1_HUMAN 8,49 12,60 no no
ROA1_HUMAN 5,32 1,57 no yes 107,108,219
ROA2_HUMAN 5,20 1,56 no yes 107,108,219
RS12_HUMAN 4,81 1,74 no no
RS13_HUMAN 5,45 1,57 no no
RS15A_HUMAN 5,70 1,66 no no
RS16_HUMAN 2,59 1,08 no no
RS19_HUMAN 2,57 1,17 no no
RS23_HUMAN 5,48 1,91 no no
RS24_HUMAN 3,12 1,64 no no
RS25_HUMAN 2,79 1,68 no no
RS26L_HUMAN 3,49 1,68 no no
RS27_HUMAN 1,45 1,67 no no
RS3A_HUMAN 2,98 1,23 no no
RS4X_HUMAN 2,43 1,00 no no
RS5_HUMAN 2,92 1,28 no no
RS7_HUMAN 3,80 1,38 no no
RS8_HUMAN 3,18 1,22 no no
RS9_HUMAN 3,51 1,29 no no
RUVB2_HUMAN 2,57 1,09 no no
SFPQ_HUMAN 6,76 1,77 no yes 107,217
Chapter 6. Appendix
176
UniProt ID -log10 (p-value) log2 FC RNP granules Stress granules Lit.
SRP09_HUMAN 2,09 1,48 no no
SRSF1_HUMAN 3,07 1,37 no no
SRSF2_HUMAN 2,79 1,23 no no
SRSF3_HUMAN 2,75 1,11 no yes 211
SRSF6_HUMAN 5,30 2,11 no no
STAU1_HUMAN 2,51 1,15 no no
SYEP_HUMAN 1,99 0,89 no no
SYNC_HUMAN 9,01 12,25 no no
TCPG_HUMAN 2,40 0,94 no no
TERA_HUMAN 2,83 1,42 no no
THIO_HUMAN 1,91 0,90 no no
TR150_HUMAN 4,04 2,86 no no
TRFL_HUMAN 1,58 1,26 no no
UBP2L_HUMAN 1,62 0,69 no yes 107–109,205,221
YBOX1_HUMAN 1,78 0,96 yes yes 107,108,116,120,206,213
Chapter 6. Appendix
6.4 Appendix - The interaction between the two protean proteins
TRPV4 and DDX3X
1,2 BMRA
BMRA + hsV4A ARD wt
hsV4A ARD wt
1,0
0,8
0,6
0,4
0,2
0,0
0 200 400 600 800
time [sec]
Figure 6.3: hsV4 ARD does not influence or inhibit the assay components lactate dehydrogenase and pyruvate kinase. As
a control for the ATPase experiments shown in section 4.2, figures 4.9 and 4.13, the well-characterized B. subtilis ATPase BmrA
was also supplemented with hsV4 ARD to rule out unspecific effects of hsV4 ARD on the lactate dehydrogenase and pyruvate
kinase used in this assay. No decrease in BmrA ATPase activity was observed upon hsV4 ARD wt supplementation. Furthermore,
hsV4 ARD alone did not show ATPase activity. BmrA was kindly provided by M. Sc.
177
Extinction [a.u.]
Chapter 6. Appendix
6.5 Appendix - An in cellulo study of the interaction between
human TRPV4 and PACSIN1-3
Sequence alignment between rat and human TRPV4 performed with ClustalW.2
sp|Q9HBA0|TRPV4_HUMAN MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRPAGP 60
sp|Q9ERZ8|TRPV4_RAT MADPGDGPRAAPGDVAEPPGDESGTSGGEAFPLSSLANLFEGEEGSSSLSPVDASRPAGP 60
*** .:****.**:*** ******* *****************:** * **.********
sp|Q9HBA0|TRPV4_HUMAN GDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDN 120
sp|Q9ERZ8|TRPV4_RAT GDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHPSDN 120
******************************************************** ***
sp|Q9HBA0|TRPV4_HUMAN KRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRL 180
sp|Q9ERZ8|TRPV4_RAT KRWRRKVVEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLSYLLTHKKRL 180
****:*::****************************************** :********
sp|Q9HBA0|TRPV4_HUMAN TDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQT 240
sp|Q9ERZ8|TRPV4_RAT TDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQT 240
************************************************************
sp|Q9HBA0|TRPV4_HUMAN ALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHI 300
sp|Q9ERZ8|TRPV4_RAT ALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHI 300
************************************************************
sp|Q9HBA0|TRPV4_HUMAN VNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDS 360
sp|Q9ERZ8|TRPV4_RAT VNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCSRLFPDS 360
*****************************************************:******
sp|Q9HBA0|TRPV4_HUMAN NLEAVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYD 420
sp|Q9ERZ8|TRPV4_RAT NLETVLNNDGLSPLMMAAKTGKIGVFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYD 420
***:********************:***********************************
sp|Q9HBA0|TRPV4_HUMAN LSSLDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLC 480
sp|Q9ERZ8|TRPV4_RAT LSSLDTCGEEVSVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLC 480
**********.*************************************************
sp|Q9HBA0|TRPV4_HUMAN AMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGV 540
sp|Q9ERZ8|TRPV4_RAT AMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLLTGVLFFFTSIKDLFMKKCPGV 540
**************************************:********.************
sp|Q9HBA0|TRPV4_HUMAN NSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTG 600
sp|Q9ERZ8|TRPV4_RAT NSLFVDGSFQLLYFIYSVLVVVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTG 600
****:***************:***************************************
sp|Q9HBA0|TRPV4_HUMAN TYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSC 660
sp|Q9ERZ8|TRPV4_RAT TYSIMIQKILFKDLFRFLLVYLLFMIGYASALVTLLNPCTNMKVCNEDQSNCTVPSYPAC 660
178
Chapter 6. Appendix
*********************************:*****:*********:*****:**:*
sp|Q9HBA0|TRPV4_HUMAN RDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGE 720
sp|Q9ERZ8|TRPV4_RAT RDSETFSAFLLDLFKLTIGMGDLEMLSSAKYPVVFILLLVTYIILTFVLLLNMLIALMGE 720
*******:********************:*******:***********************
sp|Q9HBA0|TRPV4_HUMAN TVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRV 780
sp|Q9ERZ8|TRPV4_RAT TVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRV 780
************************************************************
sp|Q9HBA0|TRPV4_HUMAN DEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDE 840
sp|Q9ERZ8|TRPV4_RAT DEVNWSHWNQNLGIINEDPGKSEIYQYYGFSHTMGRLRRDRWSSVVPRVVELNKNSGTDE 840
*********************.* *********:**********************. **
sp|Q9HBA0|TRPV4_HUMAN VVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL 871
sp|Q9ERZ8|TRPV4_RAT VVVPLDNLGNPNCDGHQQGYAPKWRAEDAPL 871
******.:***.******** ***::****
Percent Identity Matrix
1: sp|Q9HBA0|TRPV4_HUMAN 100.00 94.83
2: sp|Q9ERZ8|TRPV4_RAT 94.83 100.00
Sequence alignment between mouse and human PACSIN orthologues performed with ClustalW.2
sp|Q9BY11|PACN1_HUMAN MSSSYDEASLAPEETTDSFWEVGNYKRTVKRIDDGHRLCNDLMNCVQERAKIEKAYGQQL 60
sp|Q61644|PACN1_MOUSE MSGSYDEA---SEEITDSFWEVGNYKRTVKRIDDGHRLCNDLMSCVQERAKIEKAYAQQL 57
**.***** ** ****************************.************.***
sp|Q9BY11|PACN1_HUMAN TDWAKRWRQLIEKGPQYGSLERAWGAIMTEADKVSELHQEVKNNLLNEDLEKVKNWQKDA 120
sp|Q61644|PACN1_MOUSE TDWAKRWRQLIEKGPQYGSLERAWGAMMTEADKVSELHQEVKNSLLNEDLEKVKNWQKDA 117
**************************:****************.****************
sp|Q9BY11|PACN1_HUMAN YHKQIMGGFKETKEAEDGFRKAQKPWAKKMKELEAAKKAYHLACKEEKLAMTREMNSKTE 180
sp|Q61644|PACN1_MOUSE YHKQIMGGFKETKEAEDGFRKAQKPWAKKMKELEAAKKAYHLACKEERLAMTREMNSKTE 177
***********************************************:************
sp|Q9BY11|PACN1_HUMAN QSVTPEQQKKLQDKVDKCKQDVQKTQEKYEKVLEDVGKTTPQYMENMEQVFEQCQQFEEK 240
sp|Q61644|PACN1_MOUSE QSVTPEQQKKLVDKVDKCRQDVQKTQEKYEKVLEDVGKTTPQYMEGMEQVFEQCQQFEEK 237
*********** ******:**************************.**************
sp|Q9BY11|PACN1_HUMAN RLVFLKEVLLDIKRHLNLAENSSYIHVYRELEQAIRGADAQEDLRWFRSTSGPGMPMNWP 300
sp|Q61644|PACN1_MOUSE RLVFLKEVLLDIKRHLNLAENSSYMHVYRELEQAIRGADAQEDLRWFRSTSGPGMPMNWP 297
************************:***********************************
sp|Q9BY11|PACN1_HUMAN QFEEWNPDLPHTTTKKEKQPKKAEGVALTNATGAVESTSQAGDRGSVSSYDRGQPYATEW 360
sp|Q61644|PACN1_MOUSE QFEEWNPDLPHTTAKKEKQPKKAEGATLSNATGAVESTSQAGDRGSVSSYDRGQTYATEW 357
*************:***********.:*:************************* *****
179
Chapter 6. Appendix
sp|Q9BY11|PACN1_HUMAN SDDESGNPFGGSETNGGANPFEDDSKGVRVRALYDYDGQEQDELSFKAGDELTKLGEEDE 420
sp|Q61644|PACN1_MOUSE SDDESGNPFGGNEANGGANPFEDDAKGVRVRALYDYDGQEQDELSFKAGDELTKLGEEDE 417
***********.*:**********:***********************************
sp|Q9BY11|PACN1_HUMAN QGWCRGRLDSGQLGLYPANYVEAI 444
sp|Q61644|PACN1_MOUSE QGWCRGRLDSGQLGLYPANYVEAI 441
************************
Percent Identity Matrix
1: sp|Q9BY11|PACN1_HUMAN 100.00 95.46
2: sp|Q61644|PACN1_MOUSE 95.46 100.00
sp|Q9UNF0|PACN2_HUMAN MSVTYDDSVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCSDLMNCLHERARIEKAYAQQLT 60
sp|Q9WVE8|PACN2_MOUSE MSVTYDDSVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCGDLMNCLHERARIEKAYAQQLT 60
**************************************.*********************
sp|Q9UNF0|PACN2_HUMAN EWARRWRQLVEKGPQYGTVEKAWMAFMSEAERVSELHLEVKASLMNDDFEKIKNWQKEAF 120
sp|Q9WVE8|PACN2_MOUSE EWARRWRQLVEKGPQYGTVEKAWIAVMSEAERVSELHLEVKASLMNEDFEKIKNWQKEAF 120
***********************:*.********************:*************
sp|Q9UNF0|PACN2_HUMAN HKQMMGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHAACKEEKLAISREANSKADP 180
sp|Q9WVE8|PACN2_MOUSE HKQMMGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHTACKEEKLAISREANSKADP 180
****************************************:*******************
sp|Q9UNF0|PACN2_HUMAN SLNPEQLKKLQDKIEKCKQDVLKTKEKYEKSLKELDQGTPQYMENMEQVFEQCQQFEEKR 240
sp|Q9WVE8|PACN2_MOUSE SLNPEQLKKLQDKIEKCKQDVLKTKDKYEKSLKELDQTTPQYMENMEQVFEQCQQFEEKR 240
*************************:*********** **********************
sp|Q9UNF0|PACN2_HUMAN LRFFREVLLEVQKHLDLSNVAGYKAIYHDLEQSIRAADAVEDLRWFRANHGPGMAMNWPQ 300
sp|Q9WVE8|PACN2_MOUSE LRFFREVLLEVQKHLDLSNVASYKTIYRELEQSIKAADAVEDLRWFRANHGPGMAMNWPQ 300
*********************.**:**::*****:*************************
sp|Q9UNF0|PACN2_HUMAN FEEWSADLNRTLSRREKKKATDGVTLTGINQTGDQSLPSKPSSTLNVPSNPAQSAQSQSS 360
sp|Q9WVE8|PACN2_MOUSE FEEWSADLNRTLSRREKKKAVDGVTLTGINQTGDQSGQNKPGSNLSVPSNPAQSTQLQSS 360
********************.*************** .**.*.*.********:* ***
sp|Q9UNF0|PACN2_HUMAN YNPFEDEDDTGSTVSEKDDTKAKNVSSYEKTQSYPTDWSDDESNNPFSSTDANGDSNPFD 420
sp|Q9WVE8|PACN2_MOUSE YNPFEDEDDTGSSISEKEDIKAKNVSSYEKTQTYPTDWSDDESNNPFSSTDANGDSNPFD 420
************::***:* ************:***************************
sp|Q9UNF0|PACN2_HUMAN DDATSGTEVRVRALYDYEGQEHDELSFKAGDELTKMEDEDEQGWCKGRLDNGQVGLYPAN 480
sp|Q9WVE8|PACN2_MOUSE EDTTSGTEVRVRALYDYEGQEHDELSFKAGDELTKIEDEDEQGWCKGRLDSGQVGLYPAN 480
:*:********************************:**************.*********
sp|Q9UNF0|PACN2_HUMAN YVEAIQ 486
sp|Q9WVE8|PACN2_MOUSE YVEAIQ 486
180
Chapter 6. Appendix
******
Percent Identity Matrix
1: sp|Q9UNF0|PACN2_HUMAN 100.00 93.83
2: sp|Q9WVE8|PACN2_MOUSE 93.83 100.00
sp|Q9UKS6|PACN3_HUMAN MAPEEDAGGEALGGSFWEAGNYRRTVQRVEDGHRLCGDLVSCFQERARIEKAYAQQLADW 60
sp|Q99JB8|PACN3_MOUSE MAPEEDAGGEVLGGSFWEAGNYRRTVQRVEDGHRLCGDLVSCFQERARIEKAYAQQLADW 60
**********.*************************************************
sp|Q9UKS6|PACN3_HUMAN ARKWRGTVEKGPQYGTLEKAWHAFFTAAERLSALHLEVREKLQGQDSERVRAWQRGAFHR 120
sp|Q99JB8|PACN3_MOUSE ARKWRGAVEKGPQYGTLEKAWHAFFTAAERLSELHLEVREKLHGPDSERVRTWQRGAFHR 120
******:************************* *********:* ******:********
sp|Q9UKS6|PACN3_HUMAN PVLGGFRESRAAEDGFRKAQKPWLKRLKEVEASKKSYHAARKDEKTAQTRESHAKADSAV 180
sp|Q99JB8|PACN3_MOUSE PVLGGFRESRAAEDGFRKAQKPWLKRLKEVEASKKSYHTARKDEKTAQTRESHAKADSSM 180
**************************************:*******************::
sp|Q9UKS6|PACN3_HUMAN SQEQLRKLQERVERCAKEAEKTKAQYEQTLAELHRYTPRYMEDMEQAFETCQAAERQRLL 240
sp|Q99JB8|PACN3_MOUSE SQEQLRKLQERVGRCTKEAEKMKTQYEQTLAELNRYTPRYMEDMEQAFESCQAAERQRLL 240
************ **:***** *:*********:***************:**********
sp|Q9UKS6|PACN3_HUMAN FFKDMLLTLHQHLDLSSSEKFHELHRDLHQGIEAASDEEDLRWWRSTHGPGMAMNWPQFE 300
sp|Q99JB8|PACN3_MOUSE FFKDVLLTLHQHLDLSSSDKFHELHRDLQQSIEAASDEEDLRWWRSTHGPGMAMNWPQFE 300
****:*************:*********:*.*****************************
sp|Q9UKS6|PACN3_HUMAN EWSLDTQRTISRKEKGGRSPDEVTLTSIVPTRDGTAPPPQSPGSPGTGQDEEWSDEESPR 360
sp|Q99JB8|PACN3_MOUSE EWSLDTQRAISRKEKGGRSPDEVTLTSIVPTRDGTAPPPQSPSSPGSGQDEDWSDEESPR 360
********:*********************************.***:****:********
sp|Q9UKS6|PACN3_HUMAN KAATGVRVRALYDYAGQEADELSFRAGEELLKMSEEDEQGWCQGQLQSGRIGLYPANYVE 420
sp|Q99JB8|PACN3_MOUSE KVATGVRVRALYDYAGQEADELSFRAGEELLKMSEEDEQGWCQGQLQSGRIGLYPANYVE 420
*.**********************************************************
sp|Q9UKS6|PACN3_HUMAN CVGA 424
sp|Q99JB8|PACN3_MOUSE CVGA 424
****
Percent Identity Matrix
1: sp|Q9UKS6|PACN3_HUMAN 100.00 94.34
2: sp|Q99JB8|PACN3_MOUSE 94.34 100.00
Multiple sequence alignment between F-BARs of human PACSIN1-3 performed with ClustalW.2
sp|Q9UKS6|PACN3_F-BAR_HUMAN ---MAPEEDAGGEALGGSFWEAGNYRRTVQRVEDGHRLCGDLVSCFQERARIEKAYAQQL 57
sp|Q9BY11|PACN1_F-BAR_HUMAN MSSSYDEASLAPEETTDSFWEVGNYKRTVKRIDDGHRLCNDLMNCVQERAKIEKAYGQQL 60
181
Chapter 6. Appendix
sp|Q9UNF0|PACN2_F-BAR_HUMAN MSVTYDD-SVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCSDLMNCLHERARIEKAYAQQL 59
: . . * .****.***:***:*::******.**:.*.:***:*****.***
sp|Q9UKS6|PACN3_F-BAR_HUMAN ADWARKWRGTVEKGPQYGTLEKAWHAFFTAAERLSALHLEVREKLQGQDSERVRAWQRGA 117
sp|Q9BY11|PACN1_F-BAR_HUMAN TDWAKRWRQLIEKGPQYGSLERAWGAIMTEADKVSELHQEVKNNLLNEDLEKVKNWQKDA 120
sp|Q9UNF0|PACN2_F-BAR_HUMAN TEWARRWRQLVEKGPQYGTVEKAWMAFMSEAERVSELHLEVKASLMNDDFEKIKNWQKEA 119
::**::** :*******::*:** *::: *:::* ** **: .* .:* *::: **: *
sp|Q9UKS6|PACN3_F-BAR_HUMAN FHRPVLGGFRESRAAEDGFRKAQKPWLKRLKEVEASKKSYHAARKDEKTAQTRESHAKAD 177
sp|Q9BY11|PACN1_F-BAR_HUMAN YHKQIMGGFKETKEAEDGFRKAQKPWAKKMKELEAAKKAYHLACKEEKLAMTREMNSKTE 180
sp|Q9UNF0|PACN2_F-BAR_HUMAN FHKQMMGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHAACKEEKLAISREANSKAD 179
:*: ::***:*:: ************ *::**:**:**::* * *:** * :** ::*::
sp|Q9UKS6|PACN3_F-BAR_HUMAN SAVSQEQLRKLQERVERCAKEAEKTKAQYEQTLAELHRYTPRYMEDMEQAFETCQAAERQ 237
sp|Q9BY11|PACN1_F-BAR_HUMAN QSVTPEQQKKLQDKVDKCKQDVQKTQEKYEKVLEDVGKTTPQYMENMEQVFEQCQQFEEK 240
sp|Q9UNF0|PACN2_F-BAR_HUMAN PSLNPEQLKKLQDKIEKCKQDVLKTKEKYEKSLKELDQGTPQYMENMEQVFEQCQQFEEK 239
::. ** :***:::::* ::. **: :**: * :: : **:***:***.** ** *.:
sp|Q9UKS6|PACN3_F-BAR_HUMAN RLLFFKDMLLTLHQHLDLSSSEKFHELHRDLHQGIEAASDEED 280
sp|Q9BY11|PACN1_F-BAR_HUMAN RLVFLKEVLLDIKRHLNLAENSSYIHVYRELEQAIRGADAQED 283
sp|Q9UNF0|PACN2_F-BAR_HUMAN RLRFFREVLLEVQKHLDLSNVAGYKAIYHDLEQSIRAADAVED 282
** *::::** :::**:*:. : ::::*.*.*..*. **
Percent Identity Matrix
1: sp|Q9UKS6|PACN3_HUMAN 100.00 50.71 56.27
2: sp|Q9BY11|PACN1_HUMAN 50.71 100.00 71.63
3: sp|Q9UNF0|PACN2_HUMAN 56.27 71.63 100.00
182
Chapter 6. Appendix
6.6 Appendix -The small GTPase RhoA interacts with the TRPV4
Ankyrin Repeat Domain
15
N d
RhoA G155
[ppm]
RhoA + hsV4 ARD
G50
T163
G14 G144
I151
G17
G152
S88
S160 N123
G116
L22 N94
T60
F106
S85 S73 Q29
K135
C159 T175
T100 K162
D120 L72
E142 C16D87 N149 H105 E97
G166 K119 Q136 M134 E102
K104 L179 F171Y66
N94NH2 V33 E158D76 V11 V103 V79 K98 T77
S91
K27
E169 C107 L121 D28D90 T127 K164 R122
V38 Q180
R168 E137 A174 D146 Y74
R128
M173 K133
E143
H126
L131 I112
E93 D67
E64 A61
W99
Y156
L21
E130 R176 Y34 V167
C20 D65 D45 A177 M147
A148 D124
R129 K51
V170 R145D78 D13 Q52
I46 M1 V110A153 A132 D165
I4
L57 V139 R150
A15 K6
V48 A3
K7 F154
I95
D59 L92
D49 E172 Y42
L81 E40
A2 I10 V9V53
E47 V43
I113 E54
M82
V35
W58ind W58 I80
L55 V115
F30 R5
E125
A56
K140 L114
W99ind A44
L8
A161
1
10 8 6 H d [ppm]
Figure 6.4: 2D 1H-15N-NMR spectrum of 15N-RhoA: Overlay of spectra of 1H-15N RhoA 2D HSCQs of 15N-RhoA on its own
(grey) confirms presence of a well-folded protein in the GDP-bound state. In the presence of hsV4 ARD wt (blue), significant line
broadening is observed indicative of the formation of a high molecular weight complex. RhoA backbone NMR assignments were
transferred from previously published data.170
183
135 130 125 120 115 110 105
Chapter 6. Appendix
15
N d
RhoA G155
[ppm]
RhoA + hsV4 ARD R269C
G50
T163
G14 G144
I151
G17
G152
S88
S160 N123
G116
L22 N94
T60
S73 F106S85 Q29
K135
C159 T175
T100 K162
D120 L72
E142 C16D87 N149 H105 E97
G166 K119 M134 F171 E102K104 Q136 L179Y66
N94NH2 V33 E158D76 V11 V103 V79 K98 T77
S91
K27
E169
D90 C107 L121
D28
T127 K164 R122
V38 Q180
R168 E137 A174 D146 Y74
R128 E143
M173 K133
H126
L131 I112
E93 D67
W99
E64 A61 Y156L21
E130 V167R176 Y34
C20 D65 D45 A177 M147
A148 D124
R129 K51 R145
D78 D13 V170Q52
I46 M1 V110A153 A132 D165
I4
L57 V139 R150
A15 K6
V48
F154 I95
A3
K7 D59 L92
D49 E172 Y42
L81 E40
A2 V9
V53 I10
E47 V43
I113 E54
M82
I80 V35W58ind W58
L55 V115
F30 R5
E125
A56
K140 L114
W99ind A44
L8
A161
1
10 8 6 H d [ppm]
Figure 6.5: 2D 1H-15N-NMR spectrum of 15N-RhoA: Overlay of spectra of 1H-15N RhoA 2D HSCQs of 15N-RhoA on its own (grey)
confirms presence of a well-folded protein in the GDP-bound state. Addition of hsV4 ARD R269C (red) shows only minor effects
on the RhoA spectrum. RhoA backbone NMR assignments were transferred from previously published data.170
6.7 Appendix - Interaction of ITCH requires the full TRPV4
N-terminus in vitro
Following table contains MS data of ubiquitinated hsV4N after an in vitro ubiquitination assay with ITCH (see section 4.5, figure
4.25). MS experiments were carried out by the Johns Hopkins Mass Spectrometry and Proteomics Facility.
184
135 130 125 120 115 110 105
Chapter 6. Appendix
185
Table 6.11: MS data of ubiquitinated hsV4N after in vitro ubiquitination assay with ITCH (see section 4.5, figure 4.25). Ubiquitinated sequences
are indicated in blue row color. # PSMs = peptide spectrum matches, total number of identified peptide sequences.
Annotated Sequence Modifications # Proteins # PSMs Positions in master proteins
(R).MKFQGAFR.(K) 1xOxidation [M1] 1 5 Q9HBA0 (69-76)
(R).LTDEEFR.(E) 1 36 Q9HBA0 (180-186)
(K).RLTDEEFR.(E) 2 52 Q9HBA0 (179-186)
(R).PAGPGDGRPNLR.(M) 2 182 Q9HBA0 (57-68)
(R).EFINSPFRDIYYRGQTALHIAIER.(R) 2 4 Q9HBA0 (225-248)
(K).IIEKQPQSPK.(A) 1 2 Q9HBA0 (127-136)
(K).TGKIGIFQHIIRR.(E) 2 43 Q9HBA0 (380-392)
(K).DEGGYFYFGELPLSLAACTNQ 1xMethylThiol (C18); 1xMethylthio (C18) 2 11 Q9HBA0 (277-310)
PHIVNYLTENPHK.(K)
(R).CKHYVELLVAQGADVHAQAR.(G) 1xMethylthio (C1) 1 39 Q9HBA0 (250-269)
(R).GRFFQPK.(D) 2 13 Q9HBA0 (270-276)
(K).QPQSPK.(A) 2 5 Q9HBA0 (131-136)
(K).MYDLLLLK.(C) 1 19 Q9HBA0 (345-352)
(K).FQGAFR.(K) 2 8 Q9HBA0 (71-76)
(R).LFPDSNLEAVLNNDGLSPLMMAAK.(T) 2 26 Q9HBA0 (356-379)
(K).GVPNPIDLLESTLYESSVVPGPKK.(A) 1 13 Q9HBA0 (78-101)
(R).LFPDSNLEAVLNNDGLSPLMMAAK.(T) 1xDeamidated (N); 1xOxidation (M) 1 38 Q9HBA0 (356-379)
(R).PILFDIVSR.(G) 2 32 Q9HBA0 (152-160)
(R).ENTKFVTKMYDLLLLK.(C) 1xOxidation (M9) 1 1 Q9HBA0 (337-352)
(R).FFQPK.(D) 2 2 Q9HBA0 (272-276)
(K).IGIFQHIIRR.(E) 2 38 Q9HBA0 (383-392)
(R).LFPDSNLEAVLNNDGLSPLMMAAK.(T) 1xDeamidated (N); 2xOxidation (M20; M21) 1 95 Q9HBA0 (356-379)
Chapter 6. Appendix
186
Annotated Sequence Modifications # Proteins # PSMs Positions in master proteins
(K).GVPNPIDLLESTLYESSVVPGPK.(K) 2 53 Q9HBA0 (78-100)
(R).LFPDSNLEAVLNNDGLSPLMMAAK.(T) 2xOxidation (M20; M21) 1 276 Q9HBA0 (356-379)
(K).QPQSPKAPAPQPPPILK.(V) 2 7 Q9HBA0 (131-147)
(K).VFNRPILFDIVSR.(G) 2 206 Q9HBA0 (148-160)
(K).ALLNLSNGR.(N) 1xDeamidated (N7) 1 79 Q9HBA0 (198-206)
(R).NDTIPVLLDIAER.(T) 1xDeamidated (N1) 1 47 Q9HBA0 (207-219)
(R).GNTVLHALVAIADNTR.(E) 1xDeamidated (N) 1 76 Q9HBA0 (321-336)
(K).ALLNLSNGR.(N) 1 42 Q9HBA0 (198-206)
(R).NDTIPVLLDIAER.(T) 1 50 Q9HBA0 (207-219)
(R).GNTVLHALVAIADNTR.(E) 1 177 Q9HBA0 (321-336)
(K).ALLNLSNGR.(N) 2xDeamidated (N4; N7) 1 18 Q9HBA0 (198-206)
(K).KAPMDSLFDYGTYR.(H) 1 51 Q9HBA0 (101-114)
(K).KAPMDSLFDYGTYR.(H) 1xOxidation (M4) 1 467 Q9HBA0 (101-114)
(KR).GQTALHIAIER.(R) 3 153 Q9HBA0 (238-248)
(K).APAPQPPPILKVFNRPILFDIVSR.(G) 2 1 Q9HBA0 (137-160)
(K).HYVELLVAQGADVHAQAR.(G) 2 2595 Q9HBA0 (252-269)
(K).IGIFQHIIR.(R) 1 115 Q9HBA0 (383-391)
(R).EFINSPFR.(D) 2 62 Q9HBA0 (225-232)
(R).TGNMREFINSPFR.(D) 1xOxidation (M4) 1 3 Q9HBA0 (220-232)
(K).MYDLLLLK.(C) 1xOxidation (M1) 1 141 Q9HBA0 (345-352)
(K).APMDSLFDYGTYR.(H) 1xOxidation (M3) 1 166 Q9HBA0 (102-114)
(R).QDSRGNTVLHALVAIADNTRENTK.(F) 1 15 Q9HBA0 (317-340)
(K).VFNRPILFDIVSR.(G) 1xDeamidated (N3) 1 20 Q9HBA0 (148-160)
(R).LFPDSNLEAVLNNDGLSPLMMAAK.(T) 1xOxidation (M) 1 135 Q9HBA0 (356-379)
Chapter 6. Appendix
187
Annotated Sequence Modifications # Proteins # PSMs Positions in master proteins
(R).GSTADLDGLLPFLLTHKK.(R) 2 5 Q9HBA0 (161-178)
(K).TGKIGIFQHIIR.(R) 1 13 Q9HBA0 (380-391)
(KR).GQTALHIAIERR.(NC) 3 68 Q9HBA0 (238-249)
(R).AGPGEVAELPGDESGTPGGEAF 2 25 Q9HBA0 (10-56)
PLSSLANLFEGEDGSLSPSPADASR.(P)
(R).EFINSPFRDIYYR.(G) 2 3 Q9HBA0 (225-237)
(R).EFINSPFR.(D) 1xDeamidated (N4) 1 42 Q9HBA0 (225-232)
(R).HHSSDNK.(R) 2 5 Q9HBA0 (115-121)
(K).ALLNLSNGRNDTIPVLLDIAER.(T) 1xDeamidated (N) 1 46 Q9HBA0 (198-219)
(K).APAPQPPPILK.(V) 1 128 Q9HBA0 (137-147)
(K).APAPQPPPILK.(V) 1xDeamidated (Q5) 1 8 Q9HBA0 (137-147)
(K).RLTDEEFREPSTGK.(T) 2 96 Q9HBA0 (179-192)
(R).LTDEEFREPSTGK.(T) 2 82 Q9HBA0 (180-192)
(R).QDSRGNTVLHALVAIADNTR.(E) 1 19 Q9HBA0 (317-336)
(R).GNTVLHALVAIADNTRENTK.(F) 1 11 Q9HBA0 (321-340)
(R).GSTADLDGLLPFLLTHK.(K) 1 96 Q9HBA0 (161-177)
(R).GNTVLHALVAIADNTRENTKFVTK.(M) 1 13 Q9HBA0 (321-344)
(R).LFPDSNLEAVLNNDGLSPLMMAAKTGK.(I) 2xOxidation (M20; M21) 1 6 Q9HBA0 (356-382)
(R).RQDSRGNTVLHALVAIADNTR.(E) 1 1 Q9HBA0 (316-336)
(M).ADSSEGPR.(A) 1 13 Q9HBA0 (2-9)
(K).DEGGYFYFGELPLSLAACTNQPHI 1xMethylthio (C18) 1 4 Q9HBA0 (277-311)
VNYLTENPHKK.(A)
(K).GVPNPIDLLESTLYESSVVPGPKK.(A) 1xGG (K) 1 10 Q9HBA0 (78-101)
(K).IIEKQPQSPK.(A) 1xGG (K4) 1 1 Q9HBA0 (127-136)
Chapter 6. Appendix
188
Annotated Sequence Modifications # Proteins # PSMs Positions in master proteins
(K).KAPMDSLFDYGTYR.(H) 1xOxidation (M4); 1xGG (K1) 1 6 Q9HBA0 (101-114)
(R).AGPGEVAELPGDESGTPGGEA 2 1 Q9HBA0 (10-56)
FPLSSLANLFEGEDGSLSPSPADASR.(P)
(R).KGVPNPIDLLESTLYESSVVPGPK.(K) 1 69 Q9HBA0 (77-100)
(K).QPQSPKAPAPQPPPILK.(V) 1xGlyGly (K6); 1xGG (K6) 2 2 Q9HBA0 (131-147)
(R).KGVPNPIDLLESTLYESSVVPGPK.(K) 1xGG (K1) 1 9 Q9HBA0 (77-100)
Chapter 6. Appendix
R RR
PR P
+ +
D RD + + RDR + D GS
T
H
4N
A A H
4 M C N C
A H AR +
CH sV sV sV
4
ST I
T V4 IT V4 IT
C 4
T h h h G [kDa] hs hs sV TC
H
I h I
250
150 S3
100 S4
S1 50 S5
S2
Pure proteins Protein mixtures
Figure 6.6: Annotated BN PAGE samples submitted for MS measurements. See tables 6.12 and 6.12 for MS data.
Tables 6.12 and 6.12 show MS data of BN PAGE complexes of ITCH and hsV4N (see section 4.5, figure 4.24 ). For sample
annotation, see figure 6.6. MS experiments were carried out by the Johns Hopkins Mass Spectrometry and Proteomics Facility. Due
to the omission of detergent in the BN PAGE procedure, protein mixtures lead to smearing and therefore also slight contaminations
in bands which supposedly should only contain one protein.85 This is also reflected in the quantitative values shown in tables 6.12
and 6.12.
189
Chapter 6. Appendix
190
Table 6.12: MS data of BN PAGE complexes of ITCH and hsV4N (see section 4.5, figure 4.24 and figure 6.6 for sample annotation). Indicated
are quantitative values normalized to total MS spectra.
Accession Number Molecular Weight S1_NtermMono S1_NtermMono S2_ARD_mono S2_ARD_mono
TRPV4_HUMAN 98 kDa 294,96 301,25 361,61 367,44
ITCH_HUMAN 103 kDa 0 0 0 0
K2C1_HUMAN 66 kDa 34,829 30,233 11,533 13,42
K1C10_HUMAN 59 kDa 25,033 21,595 10,855 10,224
K1C9_HUMAN 62 kDa 20,68 24,834 8,8197 5,1122
K22E_HUMAN 65 kDa 17,414 15,116 10,177 10,864
ALBU_HUMAN 69 kDa 0 0 0 0
K1C16_HUMAN 51 kDa 8,7072 8,6379 0 0
K1C14_HUMAN 52 kDa 5,442 5,3987 4,0706 0
K2C5_HUMAN 62 kDa 0 0 0 0
K2C6A_HUMAN (+1) 60 kDa 0 0 0 0
DSG1_HUMAN 114 kDa 0 0 0 0
K1C17_HUMAN 48 kDa 0 0 0 0
IGG1_HUMAN (+1) 49 kDa 0 0 0 0
DESP_HUMAN 332 kDa 0 0 0 0
Chapter 6. Appendix
191
Accession Number Molecular Weight S3_ITCH_complex2 S3_ITCH_complex2 S4_ITCH_alone S4_ITCH_alone S5_Nterm_alone S5_Nterm_alone
TRPV4_HUMAN 98 kDa 164,67 170,2 179,41 189,14 367,54 369,73
ITCH_HUMAN 103 kDa 100,8 99,609 173,49 162,82 14,82 16,876
K2C1_HUMAN 66 kDa 31,549 32,157 15,233 18,092 11,856 7,6708
K1C10_HUMAN 59 kDa 31,549 35,294 14,387 14,802 4,9401 4,6025
K1C9_HUMAN 62 kDa 20,007 18,039 15,233 13,158 7,9041 5,1139
K22E_HUMAN 65 kDa 17,698 16,471 9,3091 6,5788 0 3,0683
ALBU_HUMAN 69 kDa 12,312 12,549 0 0 0 0
K1C16_HUMAN 51 kDa 6,9254 6,2746 0 2,467 0 0
K1C14_HUMAN 52 kDa 0 6,2746 0 0 0 0
K2C5_HUMAN 62 kDa 7,6949 7,8432 0 0 0 0
K2C6A_HUMAN (+1) 60 kDa 5,3865 0 0 0 0 0
DSG1_HUMAN 114 kDa 2,3085 0 0 0 0 0
K1C17_HUMAN 48 kDa 3,8475 0 0 0 0 0
IGG1_HUMAN (+1) 49 kDa 0 2,353 0 0 0 0
DESP_HUMAN 332 kDa 2,3085 0 0 0 0 0
Chapter 6. Appendix
6.8 Appendix - TRPV4 and the actin cytoskeleton - connecting
scientific disciplines
F-actin Merge DAPI
Figure 6.7: Fluorescence microscopy images show no effect of GSK-101 or HC-067 on the cytoskeleton of unstransfected
HEK293 cells.
192
HC-067 Temp. [37 °C] GSK-101
Chapter 6. Appendix
GFP and TRITC
GSK-101
HC-067
Temp.
0 0.2 0.4 0.6 0.8 1
r
Figure 6.8: Pearson Correlation
Coefficients (PCC, r) of respective
TRITC and DAPI
fluorescence marker pair. GFP
showed TRPV4-cGFP localization,
GSK-101
F-actin was counter-stained with
Phalloidin-TRITC and the nucleus
with DAPI. Higher PCC values
HC-067 indicate stronger linear correlations
of subjected images. See also 4.3.
Box plot whiskers indicate values
Temp. inside 1.5-fold interquartile range,
green diamonds the arithmetic mean.
Notches show 95 % confidence
0 0.2 0.4 0.6 0.8 1
r intervall of mode. Outliers are shown
as circles. nGSK-101=18, nHC-067=28,
GFP and DAPI
nTemp=33.
GSK-101
HC-067
Temp.
0 0.2 0.4 0.6 0.8 1
r
193
Chapter 6. Appendix
TRITC and DAPI
GSK-101 MTRITC
GSK-101 MDAPI
HC-067 MTRITC
Figure 6.9: Calculated M-values of
HC-067 MDAPI
respective fluorescence marker
Temp. MTRITC pairs as negative controls. GFP
showed TRPV4-cGFP localization,
Temp. MDAPI
F-actin was counter-stained with
0 20 40 60 80 100
Phalloidin-TRITC and the nucleus
%
with DAPI. For description of
M-values see equation 4.4 and
GFP and DAPI
corresponding text. Box plot whiskers
GSK-101 MGFP indicate values inside 1.5-fold in-
GSK-101 M terquartile range, green diamonds theDAPI
arithmetic mean. Notches show 95 %
HC-067 MGFP confidence intervall of mode. Outliers
HC-067 M are shown as circles. nGSK-101=18,DAPI
nHC-067=28, nTemp=33.
Temp. MGFP
Temp. MDAPI
0 20 40 60 80 100
%
Table 6.14: P and M-values of GFP (TRPV4-cGFP) and TRITC (F-actin stained with Phalloidon
TRITC) images of stably transfected HEK293 cells upon various treatments, determined and
calculated by ELSEXY (see also figures 4.28, 6.9 and 6.8). PCC = Pearson correlation coeffi-
cient r
Image IDs PCC (r) P-Value [%] MGFP-Value MTRITC-Value
GSK-101 treatment:
Aufnahme-853-Aufnahme-854 0.5200 100.00 78.2787 86.0679
Aufnahme-857-Aufnahme-858 0.2732 100.00 62.2298 81.1937
Aufnahme-861-Aufnahme-862 0.4023 100.00 5.9401 4.2444
Aufnahme-867-Aufnahme-868 0.5681 100.00 9.9422 7.3226
Aufnahme-871-Aufnahme-873 0.7425 100.00 94.2158 97.9657
Aufnahme-876-Aufnahme-877 0.8212 100.00 93.5949 97.0998
Aufnahme-880-Aufnahme-881 0.6831 100.00 95.9003 97.4444
Aufnahme-884-Aufnahme-885 0.8237 100.00 92.8449 94.4432
Aufnahme-889-Aufnahme-890 0.5636 100.00 95.5037 99.8089
Aufnahme-893-Aufnahme-894 0.7147 100.00 94.6725 98.1046
Aufnahme-897-Aufnahme-898 0.7655 100.00 92.6020 96.5671
Aufnahme-901-Aufnahme-902 0.7937 100.00 93.3543 96.6338
Aufnahme-909-Aufnahme-910 0.2758 100.00 9.6166 3.1952
Aufnahme-913-Aufnahme-914 0.2837 100.00 7.1438 3.7441
194
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MGFP-Value MTRITC-Value
Aufnahme-917-Aufnahme-918 0.2267 100.00 7.4609 2.3810
Aufnahme-921-Aufnahme-922 0.2634 100.00 69.0527 77.5663
Aufnahme-925-Aufnahme-926 0.2891 100.00 11.7675 3.1167
Aufnahme-930-Aufnahme-931 0.2809 100.00 9.1041 2.3597
Temperature treatment:
Aufnahme-715-Aufnahme-717 0.2734 100.00 56.6989 81.8237
Aufnahme-722-Aufnahme-723 0.3650 100.00 57.1273 85.8436
Aufnahme-726-Aufnahme-727 0.3448 100.00 63.5204 82.0378
Aufnahme-730-Aufnahme-731 0.4119 100.00 74.7003 92.5601
Aufnahme-734-Aufnahme-735 0.5359 100.00 84.3648 98.6884
Aufnahme-738-Aufnahme-739 0.6848 100.00 90.3637 99.3332
Aufnahme-742-Aufnahme-743 0.1857 100.00 40.4781 50.9899
Aufnahme-746-Aufnahme-747 0.4916 100.00 66.8480 83.7768
Aufnahme-750-Aufnahme-751 0.1890 100.00 48.0745 54.6190
Aufnahme-754-Aufnahme-755 0.2810 100.00 47.5923 63.3100
Aufnahme-758-Aufnahme-759 0.3313 100.00 59.1588 75.0643
Aufnahme-763-Aufnahme-764 0.5716 100.00 80.0658 91.6231
Aufnahme-768-Aufnahme-769 0.3009 100.00 54.5052 76.3846
Aufnahme-772-Aufnahme-773 0.4322 100.00 66.4405 83.4705
Aufnahme-776-Aufnahme-777 0.2188 100.00 46.8050 44.2958
Aufnahme-780-Aufnahme-781 0.3458 100.00 50.2426 54.6213
Aufnahme-784-Aufnahme-785 0.4276 100.00 76.7970 78.3063
Aufnahme-788-Aufnahme-789 0.5264 100.00 87.7442 92.1042
Aufnahme-792-Aufnahme-793 0.5648 100.00 90.8593 95.6255
Aufnahme-796-Aufnahme-797 0.1653 100.00 43.9615 63.6713
Aufnahme-800-Aufnahme-801 0.1535 100.00 38.4351 57.4055
Aufnahme-804-Aufnahme-805 0.2361 100.00 44.8965 73.5039
Aufnahme-808-Aufnahme-809 0.3448 100.00 61.8572 81.7557
Aufnahme-812-Aufnahme-813 0.4092 100.00 78.1647 93.3742
Aufnahme-816-Aufnahme-817 0.4254 100.00 78.3585 74.6014
Aufnahme-820-Aufnahme-821 0.5456 100.00 88.8685 89.4498
Aufnahme-824-Aufnahme-825 0.2165 100.00 40.9267 69.2274
Aufnahme-828-Aufnahme-829 0.2849 100.00 59.3778 79.9833
Aufnahme-837-Aufnahme-838 0.2854 100.00 64.8022 75.1479
Aufnahme-841-Aufnahme-842 0.3744 100.00 75.0187 88.7091
Aufnahme-845-Aufnahme-846 0.3607 100.00 69.7550 85.6324
Aufnahme-849-Aufnahme-850 0.4218 100.00 66.0904 84.9898
HC-067 treatment:
Aufnahme-1000-Aufnahme-1001 0.6449 100.00 92.2383 93.8185
Aufnahme-1004-Aufnahme-1005 0.6120 100.00 87.9697 89.9036
Aufnahme-1008-Aufnahme-1009 0.6150 100.00 88.5064 90.1036
Aufnahme-1012-Aufnahme-1013 0.5533 100.00 83.3883 85.0252
Aufnahme-1016-Aufnahme-1017 0.5419 100.00 81.3487 83.1448
Aufnahme-1020-Aufnahme-1021 0.5114 100.00 73.9040 77.9632
Aufnahme-1024-Aufnahme-1025 0.5629 100.00 78.7804 82.4833
Aufnahme-1028-Aufnahme-1029 0.6287 100.00 87.5814 90.4300
195
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MGFP-Value MTRITC-Value
Aufnahme-1032-Aufnahme-1033 0.6687 100.00 92.2003 93.6834
Aufnahme-1036-Aufnahme-1037 0.6806 100.00 91.7884 92.6963
Aufnahme-1040-Aufnahme-1041 0.6476 100.00 92.9055 93.5310
Aufnahme-1045-Aufnahme-1046 0.6432 100.00 88.5934 89.5814
Aufnahme-1049-Aufnahme-1050 0.7043 100.00 90.0797 91.3065
Aufnahme-1053-Aufnahme-1054 0.6106 100.00 90.8049 92.5187
Aufnahme-1057-Aufnahme-1058 0.6569 100.00 90.5583 92.7421
Aufnahme-1061-Aufnahme-1062 0.6972 100.00 91.0875 92.3391
Aufnahme-1065-Aufnahme-1066 0.6118 100.00 88.4876 89.9069
Aufnahme-1069-Aufnahme-1070 0.6212 100.00 87.1519 89.3313
Aufnahme-938-Aufnahme-939 0.6324 100.00 10.5299 3.4553
Aufnahme-942-Aufnahme-943 0.6250 100.00 85.4327 87.9572
Aufnahme-946-Aufnahme-947 0.4504 100.00 6.3986 2.6539
Aufnahme-950-Aufnahme-951 0.4398 100.00 76.8892 81.2257
Aufnahme-954-Aufnahme-955 0.6594 100.00 91.6085 92.9828
Aufnahme-958-Aufnahme-959 0.7064 100.00 94.0716 95.2790
Aufnahme-962-Aufnahme-963 0.4668 100.00 72.5181 75.0115
Aufnahme-966-Aufnahme-967 0.5041 100.00 73.5635 76.5784
Aufnahme-972-Aufnahme-973 0.5297 100.00 81.9084 84.7888
Aufnahme-976-Aufnahme-977 0.4837 100.00 79.9120 82.8985
Table 6.15: P and M-values of TRITC (F-actin stained with Phalloidon TRITC) and DAPI (Nu-
cleus) images of stably transfected HEK293 cells upon various treatments, determined and cal-
culated by ELSEXY (see also figures 4.28, 6.9 and 6.8). PCC = Pearson correlation coefficient
r
Image IDs PCC (r) P-Value [%] MTRITC-Value MDAPI-Value
GSK-101 treatment:
Aufnahme-854-Aufnahme-855 0.0453 87.75 2.2698 1.9382
Aufnahme-858-Aufnahme-859 -0.0776 99.99 9.0303 4.6092
Aufnahme-862-Aufnahme-863 -0.0823 99.99 4.7414 1.0634
Aufnahme-868-Aufnahme-869 -0.0120 53.32 2.9750 1.7098
Aufnahme-873-Aufnahme-874 -0.1359 100.00 5.9047 1.5552
Aufnahme-877-Aufnahme-878 -0.0314 72.60 6.2035 1.4343
Aufnahme-881-Aufnahme-882 -0.0318 73.16 4.9780 1.1867
Aufnahme-885-Aufnahme-887 -0.0101 52.66 5.5307 1.1056
Aufnahme-890-Aufnahme-891 -0.0088 52.05 6.6444 1.4626
Aufnahme-894-Aufnahme-895 -0.0241 62.51 4.6918 0.9261
Aufnahme-898-Aufnahme-899 0.0112 53.11 5.7763 1.3593
Aufnahme-902-Aufnahme-903 0.0100 52.55 5.2710 1.1873
Aufnahme-910-Aufnahme-911 -0.0271 67.22 6.4353 2.6085
196
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MTRITC-Value MDAPI-Value
Aufnahme-914-Aufnahme-915 -0.0308 70.34 4.9065 2.5552
Aufnahme-918-Aufnahme-919 0.0156 56.52 4.2131 1.5957
Aufnahme-922-Aufnahme-923 0.1897 100.00 3.3455 1.5303
Aufnahme-926-Aufnahme-927 0.0370 79.59 4.5315 1.7017
Aufnahme-931-Aufnahme-932 0.0225 62.21 2.7836 0.9981
Temperature treatment:
Aufnahme-715-Aufnahme-720 -0.0279 69.65 3.6567 2.1010
Aufnahme-722-Aufnahme-724 0.0025 50.14 1.2990 0.5988
Aufnahme-726-Aufnahme-728 -0.0571 96.69 4.4134 2.3618
Aufnahme-730-Aufnahme-732 -0.0304 71.63 1.6476 0.8968
Aufnahme-734-Aufnahme-736 -0.1550 100.00 3.7128 2.6887
Aufnahme-738-Aufnahme-740 -0.0339 76.10 2.5587 1.5470
Aufnahme-742-Aufnahme-744 -0.1645 100.00 9.7233 3.3940
Aufnahme-746-Aufnahme-748 -0.1533 100.00 2.7878 1.5764
Aufnahme-750-Aufnahme-752 -0.0598 98.28 0.1225 0.0027
Aufnahme-754-Aufnahme-756 -0.0191 58.85 5.3142 2.1340
Aufnahme-758-Aufnahme-760 -0.0544 96.39 4.9039 2.5055
Aufnahme-763-Aufnahme-765 -0.0743 99.98 4.2717 2.2736
Aufnahme-768-Aufnahme-770 -0.0419 86.53 12.5606 14.5611
Aufnahme-772-Aufnahme-774 -0.1626 100.00 3.7872 3.3313
Aufnahme-776-Aufnahme-778 -0.1146 100.00 5.9939 2.2270
Aufnahme-780-Aufnahme-782 -0.0635 99.43 4.8804 3.6756
Aufnahme-784-Aufnahme-786 -0.0368 79.79 11.1294 3.5519
Aufnahme-788-Aufnahme-790 -0.1036 100.00 6.3836 3.2530
Aufnahme-792-Aufnahme-794 -0.1636 100.00 3.5069 2.2702
Aufnahme-796-Aufnahme-798 0.0103 52.55 24.3181 41.1005
Aufnahme-800-Aufnahme-802 0.1172 100.00 0.1573 0.0037
Aufnahme-804-Aufnahme-806 -0.0439 88.24 7.3157 11.4028
Aufnahme-808-Aufnahme-810 -0.0128 54.17 5.7927 2.2767
Aufnahme-812-Aufnahme-814 -0.0659 99.45 3.3155 1.0491
Aufnahme-816-Aufnahme-818 0.1985 100.00 0.4610 0.0281
Aufnahme-820-Aufnahme-822 -0.1803 100.00 5.3852 2.9802
Aufnahme-824-Aufnahme-826 -0.0228 61.88 5.1411 2.5597
Aufnahme-828-Aufnahme-830 0.1261 100.00 33.0759 53.0580
Aufnahme-837-Aufnahme-839 -0.0173 56.81 11.0905 3.3428
Aufnahme-841-Aufnahme-843 -0.0255 64.93 4.7149 1.9215
Aufnahme-845-Aufnahme-847 -0.0404 83.73 6.3069 2.0690
Aufnahme-849-Aufnahme-851 -0.0426 87.13 2.9024 1.4929
HC-067:
Aufnahme-1001-Aufnahme-1002 -0.0259 67.38 1.9920 0.4166
Aufnahme-1005-Aufnahme-1006 -0.1626 100.00 2.1678 0.4999
Aufnahme-1009-Aufnahme-1010 -0.1890 100.00 1.5087 0.3787
Aufnahme-1013-Aufnahme-1014 -0.0232 64.10 3.6097 0.8075
Aufnahme-1017-Aufnahme-1018 -0.0244 67.30 2.5572 0.5483
Aufnahme-1021-Aufnahme-1022 -0.1046 100.00 8.3782 1.7364
Aufnahme-1025-Aufnahme-1026 -0.0623 99.29 7.2097 1.6761
197
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MTRITC-Value MDAPI-Value
Aufnahme-1029-Aufnahme-1030 -0.0603 98.82 11.7791 2.7133
Aufnahme-1033-Aufnahme-1034 -0.0585 98.79 5.5796 1.3661
Aufnahme-1037-Aufnahme-1038 -0.0731 99.96 4.1914 0.8995
Aufnahme-1041-Aufnahme-1042 -0.0707 99.92 5.0806 1.1388
Aufnahme-1046-Aufnahme-1047 -0.0963 100.00 7.8957 1.5447
Aufnahme-1050-Aufnahme-1051 0.0217 62.47 5.5741 1.3416
Aufnahme-1054-Aufnahme-1055 -0.0581 97.64 3.9439 0.9346
Aufnahme-1058-Aufnahme-1059 -0.1628 100.00 3.5124 0.8515
Aufnahme-1062-Aufnahme-1063 -0.0444 89.74 2.3205 0.7769
Aufnahme-1066-Aufnahme-1067 -0.0325 74.71 2.2184 0.4525
Aufnahme-1070-Aufnahme-1071 -0.0152 56.12 1.1900 0.3502
Aufnahme-939-Aufnahme-940 -0.1093 100.00 1.3381 0.3299
Aufnahme-943-Aufnahme-944 -0.1054 100.00 0.8397 0.2275
Aufnahme-947-Aufnahme-948 -0.0382 83.89 1.6123 0.4816
Aufnahme-951-Aufnahme-952 -0.1044 100.00 0.9972 0.3373
Aufnahme-955-Aufnahme-956 -0.1044 100.00 2.4847 0.6036
Aufnahme-959-Aufnahme-960 -0.0634 99.80 1.7299 0.3815
Aufnahme-963-Aufnahme-964 -0.0429 87.90 2.5499 0.7984
Aufnahme-967-Aufnahme-968 -0.1261 100.00 1.6800 0.4122
Aufnahme-973-Aufnahme-974 -0.1074 100.00 1.2766 0.1771
Aufnahme-977-Aufnahme-978 -0.1154 100.00 0.9108 0.1482
Table 6.16: P and M-values of GFP (TRPV4-cGFP) and DAPI (Nucleus) images of stably trans-
fected HEK293 cells upon various treatments, determined and calculated by ELSEXY (see also
figures 4.28, 6.9 and 6.8). PCC = Pearson correlation coefficient r
Image IDs PCC (r) P-Value [%] MGFP-Value MDAPI-Value
GSK-101 treatment:
Aufnahme-853-Aufnahme-855 -0.1403 100.00 0.0100 0.0041
Aufnahme-857-Aufnahme-859 -0.0333 76.46 0.5327 0.3642
Aufnahme-861-Aufnahme-863 -0.0847 100.00 3.2794 0.7690
Aufnahme-867-Aufnahme-869 0.0096 52.33 1.7832 0.9969
Aufnahme-871-Aufnahme-874 -0.0300 73.40 0.9859 0.4895
Aufnahme-876-Aufnahme-878 -0.0167 57.37 0.7300 0.3754
Aufnahme-880-Aufnahme-882 -0.0435 92.08 0.8073 0.3559
Aufnahme-884-Aufnahme-887 -0.0289 70.83 0.7969 0.3878
Aufnahme-889-Aufnahme-891 -0.0163 58.52 1.2954 0.7190
Aufnahme-893-Aufnahme-895 0.0280 72.08 42.9159 56.7900
Aufnahme-897-Aufnahme-899 0.0570 98.34 38.5048 52.4213
Aufnahme-901-Aufnahme-903 0.0855 100.00 49.5204 64.0538
Aufnahme-909-Aufnahme-911 0.0146 55.46 9.8164 2.9068
Aufnahme-913-Aufnahme-915 0.0367 77.81 2.8104 1.4889
198
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MGFP-Value MDAPI-Value
Aufnahme-917-Aufnahme-919 0.0218 61.57 9.1948 2.5784
Aufnahme-921-Aufnahme-923 -0.3342 100.00 0.8344 0.2907
Aufnahme-925-Aufnahme-927 0.0168 57.13 6.9142 1.1782
Aufnahme-930-Aufnahme-932 -0.0003 50.00 2.2941 0.2445
Temperature treatment:
Aufnahme-715-Aufnahme-720 -0.0279 69.65 3.6567 2.1010
Aufnahme-722-Aufnahme-724 0.0025 50.14 1.2990 0.5988
Aufnahme-726-Aufnahme-728 -0.0571 96.69 4.4134 2.3618
Aufnahme-730-Aufnahme-732 -0.0304 71.63 1.6476 0.8968
Aufnahme-734-Aufnahme-736 -0.1550 100.00 3.7128 2.6887
Aufnahme-738-Aufnahme-740 -0.0339 76.10 2.5587 1.5470
Aufnahme-742-Aufnahme-744 -0.1645 100.00 9.7233 3.3940
Aufnahme-746-Aufnahme-748 -0.1533 100.00 2.7878 1.5764
Aufnahme-750-Aufnahme-752 -0.0598 98.28 0.1225 0.0027
Aufnahme-754-Aufnahme-756 -0.0191 58.85 5.3142 2.1340
Aufnahme-758-Aufnahme-760 -0.0544 96.39 4.9039 2.5055
Aufnahme-763-Aufnahme-765 -0.0743 99.98 4.2717 2.2736
Aufnahme-768-Aufnahme-770 -0.0419 86.53 12.5606 14.5611
Aufnahme-772-Aufnahme-774 -0.1626 100.00 3.7872 3.3313
Aufnahme-776-Aufnahme-778 -0.1146 100.00 5.9939 2.2270
Aufnahme-780-Aufnahme-782 -0.0635 99.43 4.8804 3.6756
Aufnahme-784-Aufnahme-786 -0.0368 79.79 11.1294 3.5519
Aufnahme-788-Aufnahme-790 -0.1036 100.00 6.3836 3.2530
Aufnahme-792-Aufnahme-794 -0.1636 100.00 3.5069 2.2702
Aufnahme-796-Aufnahme-798 0.0103 52.55 24.3181 41.1005
Aufnahme-800-Aufnahme-802 0.1172 100.00 0.1573 0.0037
Aufnahme-804-Aufnahme-806 -0.0439 88.24 7.3157 11.4028
Aufnahme-808-Aufnahme-810 -0.0128 54.17 5.7927 2.2767
Aufnahme-812-Aufnahme-814 -0.0659 99.45 3.3155 1.0491
Aufnahme-816-Aufnahme-818 0.1985 100.00 0.4610 0.0281
Aufnahme-820-Aufnahme-822 -0.1803 100.00 5.3852 2.9802
Aufnahme-824-Aufnahme-826 -0.0228 61.88 5.1411 2.5597
Aufnahme-828-Aufnahme-830 0.1261 100.00 33.0759 53.0580
Aufnahme-837-Aufnahme-839 -0.0173 56.81 11.0905 3.3428
Aufnahme-841-Aufnahme-843 -0.0255 64.93 4.7149 1.9215
Aufnahme-845-Aufnahme-847 -0.0404 83.73 6.3069 2.0690
Aufnahme-849-Aufnahme-851 -0.0426 87.13 2.9024 1.4929
HC-067:
Aufnahme-1000-Aufnahme-1002 -0.0451 90.68 3.1690 0.5565
Aufnahme-1004-Aufnahme-1006 -0.1631 100.00 4.8925 0.8189
Aufnahme-1008-Aufnahme-1010 -0.0314 75.59 3.7647 0.5352
Aufnahme-1012-Aufnahme-1014 0.0087 52.10 8.4618 1.4623
Aufnahme-1016-Aufnahme-1018 -0.0271 68.35 6.1828 1.0086
Aufnahme-1020-Aufnahme-1022 -0.0165 56.18 13.2830 2.1062
Aufnahme-1024-Aufnahme-1026 -0.0916 100.00 10.9515 1.8920
Aufnahme-1028-Aufnahme-1030 -0.0441 87.52 13.6688 3.2934
199
Chapter 6. Appendix
Image IDs PCC (r) P-Value [%] MGFP-Value MDAPI-Value
Aufnahme-1032-Aufnahme-1034 -0.0549 96.16 7.6856 1.7986
Aufnahme-1036-Aufnahme-1038 -0.0653 99.69 7.7639 1.8226
Aufnahme-1040-Aufnahme-1042 -0.0682 99.91 8.9296 2.1581
Aufnahme-1045-Aufnahme-1047 -0.0622 99.19 10.9042 2.4098
Aufnahme-1049-Aufnahme-1051 -0.0494 93.04 7.4204 1.5090
Aufnahme-1053-Aufnahme-1055 -0.0588 98.72 6.3371 1.6009
Aufnahme-1057-Aufnahme-1059 -0.0606 99.16 4.6242 1.3025
Aufnahme-1061-Aufnahme-1063 -0.0501 95.10 4.3516 1.0979
Aufnahme-1065-Aufnahme-1067 -0.1420 100.00 7.4282 1.2888
Aufnahme-1069-Aufnahme-1071 -0.0398 84.78 3.8079 0.7245
Aufnahme-938-Aufnahme-940 -0.1324 100.00 5.5320 0.8956
Aufnahme-942-Aufnahme-944 -0.0697 99.95 2.5526 0.5187
Aufnahme-946-Aufnahme-948 -0.0264 67.77 4.6858 0.9460
Aufnahme-950-Aufnahme-952 -0.0230 63.73 2.5941 0.6412
Aufnahme-954-Aufnahme-956 -0.0467 94.75 6.3291 1.3117
Aufnahme-958-Aufnahme-960 -0.0520 96.46 4.0165 0.8191
Aufnahme-962-Aufnahme-964 -0.0228 62.86 7.9805 1.6780
Aufnahme-966-Aufnahme-968 -0.0048 50.52 5.0571 1.1735
Aufnahme-972-Aufnahme-974 0.1943 100.00 3.7948 1.2591
Aufnahme-976-Aufnahme-978 0.0681 99.70 2.8181 0.9724
200
Chapter 6. Appendix
6.9 Appendix - Establishing HEK293 cell lines stably expressing
TRP channels
In the course of this work, HEK293 cells stably expressing human TRPV4-cGFP and TRPML1-cGFP, respectively, were estab-
lished (see section 3.2.3 for procedure). In both cases, fluorescence microscopy images showed expected subcellular localization,
as shown for human TRPV4-cGFP in figure 6.10 and human TRPML1-cGFP 6.12. Furthermore, for HEK293 hsTRPV4-cGFP cells,
functionality of hsTRPV4-cGFP was confirmed via a Ca2+-influx assay, in agreement with McCray et al.69
hsTRPV4-cGFP DAPI Merge
Figure 6.10: Fluorescence microscopy images of HEK293 cells stably transfected with hsTRPV4-cGFP. Stable transfection
was achieved as described in subsection 3.2.3. hsTRPV4-cGFP shows expected plasma membrane localization.
Figure 6.11: Stably transfected HEK293 cells express funtional hsTRPV4-cGFP. Functionality of expressed hsTRPV4-cGFP
upon GSK101 activation, evaluated via a Ca2+-influx assay performed with the Fluo-4 Direct Calcium Assay (see table 2.18).
x-axis = time in seconds, y-axis: fluorescence in arbitrary units. Averages represent n = 3, error bars indicate SD.
201
fluorescence [au]
Chapter 6. Appendix
hsTRPML-1 cGFP DAPI Merge
Figure 6.12: Fluorescence microscopy images of HEK293 cells stabily transfected with hsTRPML1-cGFP. Stable trans-
fection was achieved as described in subsection 3.2.3. hsTRPML1-cGFP shows expected localization in vesicular structures
(indicated by white arrow heads), which are most likely lysosomes.229
6.10 Appendix - Purification of ITCH WW domains
ITCH contains four WW domains (WW1-4), which could be possible interaction domains for the TRPV4 PRR.230 To probe possible
PPIs between the ITCH WW domains and TRPV4 PRR in detail recombinant expression of all four single WW domains and the
tandem domains WW1+2 and 3+4 was established (see section 3.7.8). Furthermore, first purifications and characterizations of the
WW1+2 and 2+3 tandem domains were carried out (figure 6.13).
A B
5 ITCH WW1+2 tandem domain
1400 ITCH WW1+2 tandem domain
ITCH WW3+4 tandem domain
ITCH WW3+4 tandem domain
1200
MW MW
Coomassie [kDa] Coomassie [kDa] 0
1000 1 .0 random coil
50
50 b-sheet
37 0 .9 a-helix
37
0 .8
800 25 25
20 0 .7
20 -5
15 0 .6
600 15
0 .5
10
10 0 .4
400 0 .3
ITCH ITCH -10
WW1+2 WW3+4 0 .2
0 .1
200 Superdex75
0 .0
CH H
IT
2
1+ IT
C
3+
4
W W
W W
0 -15
40 50 60 70 80 90 100 110 120 190 200 210 220 230 240 250
Volume [mL] l [nm]
Figure 6.13: Purification of recombinant human ITCH WW domains A SEC purification of human ITCH WW1+2 and WW3+4
tandem domains. SEC runs were performed with a HiLoad Superdex 75 pg preparative SEC column. Inlet shows Coomassie
stained 15 % SDS-PAGE of collected and concentrated fractions after SEC. x-axis: eluted volume in mL, y-axis: absorbency
at 280 nm, M = marker. B Far UV CD spectrum of human WW1+2 and WW3+4 tandem domains. Spectra were measured at
293 K and proteins were used at 1µM concentration in either SEC Buffer with a final NaCl concentration of 20 mM NaCl. Inlet
shows secondary structure prediction performed with CAPITO.93 The predicted α-helix/β-sheet/random coil content of the purified
proteins is for ITCH WW1+2 tandem domain 10 %/32 %/58 % and ITCH WW3+4 tandem domain 5 %/37 %/59 %. x-axis: mean
residue ellipticity ([Θ] −3mrw,λ) in 10 deg cm2 dmol−1, y-axis: wavelength λ in nm.
202
Abs [mAu]
280nm
3 2 -1
q [10 deg cm dmol ]
mrw, l
sec. structure content
Chapter 6. Appendix
6.11 Appendix - Purification of YAP1 WW domains
YAP1 contains two WW domains, which could be possible interaction domains for the TRPV4 PRR.230 To probe possible PPIs
between the YAP1 WW domains and TRPV4 PRR in detail, recombinant expression of the YAP-1 WW1 domain and YAP1 WW1+2
tandem domain was established (see section 3.7.9). Furthermore, first purifications and characterizations of the YAP1 WW1 and
WW1+2 tandem domain were carried out (figure 6.14). Expression conditions and subsequent purification of the YAP1 WW2
domain have still to be optimized.
A B
1200 MW 5
[kDa]
hsYAP1 WW1+2 Coomassie hsYAP1 WW1+2
hsYAP-1 WW1 50 hsYAP1 WW1
hsYAP-1 WW2 37
1000 25
20
0
15
800 1 .0 random coil
10
b-sheet
0 .9 a-helix
YAP-1 YAP-1 YAP-1
WW1 WW2 WW1+2 0 .8
600 -5 0 .7
0 .6
0 .5
0 .4
400
0 .3
-10 0 .2
0 .1
200 Superdex75
0 .0
1 1
YA
P
+2
P
1 YA 2
W W
W W
0 -15
50 60 70 80 90 100 110 120 200 210 220 230 240 250
Volume [mL] l [nm]
Figure 6.14: Purification of recombinant human YAP1 WW domains A SEC purification of human YAP1 WW1+2 tandem and
YAP1 WW1 domain. SEC runs were performed with a HiLoad Superdex 75 pg preparative SEC column. Inlet shows Coomassie
stained 15 % SDS-PAGE of collected and concentrated fractions after SEC. x-axis: eluted volume in mL, y-axis: absorbency at
280 nm, M = marker. B Far UV CD spectrum of human YAP1 WW1+2 tandem and YAP1 WW1 domain. Spectra were measured
at 293 K and proteins were used at 1µM concentration in either SEC Buffer with a final NaCl concentration of 20 mM NaCl.
Inlet shows secondary structure prediction performed with CAPITO.93 The predicted α-helix/β-sheet/random coil content of the
purified proteins is for YAP WW1+2 tandem domain 3 %/50 %/47 % and YAP1 WW1 domain 2 %/42 %/56 %. x-axis: mean residue
ellipticity ([Θ]mrw,λ) in 10−3 deg cm2 dmol−1, y-axis: wavelength λ in nm.
203
Abs [mAu]
280nm
3 2 -1
q [10 deg cm dmol ]
mrw, l
sec. structure content
Chapter 6. Appendix
6.12 Appendix - Amino acid and DNA sequences
hsV4N
atggcggattccagcgaaggcccccgcgcggggcccggggaggtggctgagctccccggggatgagagtggcaccccaggtggggaggcttttcctctc
M A D S S E G P R A G P G E V A E L P G D E S G T P G G E A F P L
tcctccctggccaatctgtttgagggggaggatggctccctttcgccctcaccggctgatgccagtcgccctgctggcccaggcgatgggcgaccaaat
S S L A N L F E G E D G S L S P S P A D A S R P A G P G D G R P N
ctgcgcatgaagttccagggcgccttccgcaagggggtgcccaaccccatcgatctgctggagtccaccctatatgagtcctcggtggtgcctgggccc
L R M K F Q G A F R K G V P N P I D L L E S T L Y E S S V V P G P
aagaaagcacccatggactcactgtttgactacggcacctatcgtcaccactccagtgacaacaagaggtggaggaagaagatcatagagaagcagccg
K K A P M D S L F D Y G T Y R H H S S D N K R W R K K I I E K Q P
cagagccccaaagcccctgcccctcagccgccccccatcctcaaagtcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgac
Q S P K A P A P Q P P P I L K V F N R P I L F D I V S R G S T A D
ctggacgggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggcc
L D G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A
ttgctgaacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttc
L L N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F
cgtgacatctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtc
R D I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V
cacgcccaggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccag
H A Q A R G R F F Q P K D E G G Y F Y F G E L P L S L A A C T N Q
ccccacattgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggcc
P H I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A
attgctgacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggag
I A D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E
gccgtgctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggat
A V L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D
gaggcggccgctcatcatcaccatcatcattga
E A A A H H H H H H -
hsV4N wt Avi
atggcggattccagcgaaggcccccgcgcggggcccggggaggtggctgagctccccggggatgagagtggcaccccaggtggggaggcttttcctctc
M A D S S E G P R A G P G E V A E L P G D E S G T P G G E A F P L
tcctccctggccaatctgtttgagggggaggatggctccctttcgccctcaccggctgatgccagtcgccctgctggcccaggcgatgggcgaccaaat
S S L A N L F E G E D G S L S P S P A D A S R P A G P G D G R P N
ctgcgcatgaagttccagggcgccttccgcaagggggtgcccaaccccatcgatctgctggagtccaccctatatgagtcctcggtggtgcctgggccc
L R M K F Q G A F R K G V P N P I D L L E S T L Y E S S V V P G P
aagaaagcacccatggactcactgtttgactacggcacctatcgtcaccactccagtgacaacaagaggtggaggaagaagatcatagagaagcagccg
K K A P M D S L F D Y G T Y R H H S S D N K R W R K K I I E K Q P
cagagccccaaagcccctgcccctcagccgccccccatcctcaaagtcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgac
Q S P K A P A P Q P P P I L K V F N R P I L F D I V S R G S T A D
ctggacgggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggcc
L D G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A
ttgctgaacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttc
204
Chapter 6. Appendix
L L N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F
cgtgacatctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtc
R D I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V
cacgcccaggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccag
H A Q A R G R F F Q P K D E G G Y F Y F G E L P L S L A A C T N Q
ccccacattgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggcc
P H I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A
attgctgacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggag
I A D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E
gccgtgctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggat
A V L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D
gaggcggccgctggcctgaacgatatttttgaagcgcagaaaattgaatggcatgaacatcatcaccatcatcattga
E A A A G L N D I F E A Q K I E W H E H H H H H H -
hsV4 ARD wt Avi
atgcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgac
M F N R P I L F D I V S R G S T A D
ctggacgggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggcc
L D G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A
ttgctgaacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttc
L L N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F
cgtgacatctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtc
R D I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V
cacgcccaggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccag
H A Q A R G R F F Q P K D E G G Y F Y F G E L P L S L A A C T N Q
ccccacattgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggcc
P H I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A
attgctgacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggag
I A D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E
gccgtgctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggat
A V L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D
gaggcggccgctggcctgaacgatatttttgaagcgcagaaaattgaatggcatgaacatcatcaccatcatcattga
E A A A G L N D I F E A Q K I E W H E H H H H H H -
hsV4 ARD R232C Avi
atgcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgac
M F N R P I L F D I V S R G S T A D
ctggacgggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggcc
L D G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A
ttgctgaacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttc
L L N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F
tgtgacatctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtc
C D I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V
cacgcccaggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccag
205
Chapter 6. Appendix
H A Q A R G R F F Q P K D E G G Y F Y F G E L P L S L A A C T N Q
ccccacattgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggcc
P H I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A
attgctgacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggag
I A D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E
gccgtgctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggat
A V L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D
gaggcggccgctggcctgaacgatatttttgaagcgcagaaaattgaatggcatgaacatcatcaccatcatcattga
E A A A G L N D I F E A Q K I E W H E H H H H H H -
hsV4 ARD K276E Avi
atgcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgac
M F N R P I L F D I V S R G S T A D
ctggacgggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggcc
L D G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A
ttgctgaacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttc
L L N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F
cgtgacatctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtc
R D I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V
cacgcccaggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccag
H A Q A R G R F F Q P E D E G G Y F Y F G E L P L S L A A C T N Q
ccccacattgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggcc
P H I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A
attgctgacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggag
I A D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E
gccgtgctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggat
A V L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D
gaggcggccgctggcctgaacgatatttttgaagcgcagaaaattgaatggcatgaacatcatcaccatcatcattga
E A A A G L N D I F E A Q K I E W H E H H H H H H -
BirA ligase
atgaaggataacaccgtgccactgaaattgattgccctgttagcgaacggtgaatttcactctggcgagcagttgggtgaaacgctgggaatgagccgg
M K D N T V P L K L I A L L A N G E F H S G E Q L G E T L G M S R
gcggctattaataaacacattcagacactgcgtgactggggcgttgatgtctttaccgttccgggtaaaggatacagcctgcctgagcctatccagtta
A A I N K H I Q T L R D W G V D V F T V P G K G Y S L P E P I Q L
cttaatgctaaacagatattgggtcagctggatggcggtagtgtagccgtgctgccagtgattgactccacgaatcagtaccttcttgatcgtatcgga
L N A K Q I L G Q L D G G S V A V L P V I D S T N Q Y L L D R I G
gagcttaaatcgggcgatgcttgcattgcagaataccagcaggctggccgtggtcgccggggtcggaaatggttttcgccttttggcgcaaacttatat
E L K S G D A C I A E Y Q Q A G R G R R G R K W F S P F G A N L Y
ttgtcgatgttctggcgtctggaacaaggcccggcggcggcgattggtttaagtctggttatcggtatcgtgatggcggaagtattacgcaagctgggt
L S M F W R L E Q G P A A A I G L S L V I G I V M A E V L R K L G
gcagataaagttcgtgttaaatggcctaatgacctctatctgcaggatcgcaagctggcaggcattctggtggagctgactggcaaaactggcgatgcg
A D K V R V K W P N D L Y L Q D R K L A G I L V E L T G K T G D A
gcgcaaatagtcattggagccgggatcaacatggcaatgcgccgtgttgaagagagtgtcgttaatcaggggtggatcacgctgcaggaagcggggatc
206
Chapter 6. Appendix
A Q I V I G A G I N M A M R R V E E S V V N Q G W I T L Q E A G I
aatctcgatcgtaatacgttggcggccatgttaatacgtgaattacgtgctgcgttggaactcttcgaacaagaaggattggcaccttatctgtcgcgc
N L D R N T L A A M L I R E L R A A L E L F E Q E G L A P Y L S R
tgggaaaagctggataattttattaatcgcccagtgaaacttatcattggtgataaagaaatatttggcatttcacgcggaatagacaaacagggggct
W E K L D N F I N R P V K L I I G D K E I F G I S R G I D K Q G A
ttattacttgagcaggatggaataataaaaccctggatgggcggtgaaatatccctgcgtagtgcagaaaaaggtggtctcgagtga
L L L E Q D G I I K P W M G G E I S L R S A E K G G L E -
hsV4 ARD+PRR
atgaaagcccctgcccctcagccgccccccatcctcaaagtcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgacctggac
M K A P A P Q P P P I L K V F N R P I L F D I V S R G S T A D L D
gggctgctcccattcttgctgacccacaagaaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggccttgctg
G L L P F L L T H K K R L T D E E F R E P S T G K T C L P K A L L
aacctgagcaatggccgcaacgacaccatccctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttccgtgac
N L S N G R N D T I P V L L D I A E R T G N M R E F I N S P F R D
atctactatcgaggtcagacagccctgcacatcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtccacgcc
I Y Y R G Q T A L H I A I E R R C K H Y V E L L V A Q G A D V H A
caggcccgtgggcgcttcttccagcccaaggatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccagccccac
Q A R G R F F Q P K D E G G Y F Y F G E L P L S L A A C T N Q P H
attgtcaactacctgacggagaacccccacaagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggccattgct
I V N Y L T E N P H K K A D M R R Q D S R G N T V L H A L V A I A
gacaacacccgtgagaacaccaagtttgttaccaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggaggccgtg
D N T R E N T K F V T K M Y D L L L L K C A R L F P D S N L E A V
ctcaacaacgacggcctctcgcccctcatgatggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggatgaggcg
L N N D G L S P L M M A A K T G K I G I F Q H I I R R E V T D E A
gccgctcatcatcaccatcatcattga
A A H H H H H H -
hsV4 ARD wt
atgctcaaagtcttcaaccggcctatcctctttgacatcgtgtcccggggctccactgctgacctggacgggctgctcccattcttgctgacccacaag
M L K V F N R P I L F D I V S R G S T A D L D G L L P F L L T H K
aaacgcctaactgatgaggagtttcgagagccatctacggggaagacctgcctgcccaaggccttgctgaacctgagcaatggccgcaacgacaccatc
K R L T D E E F R E P S T G K T C L P K A L L N L S N G R N D T I
cctgtgctgctggacatcgcggagcgcaccggcaacatgagggagttcattaactcgcccttccgtgacatctactatcgaggtcagacagccctgcac
P V L L D I A E R T G N M R E F I N S P F R D I Y Y R G Q T A L H
atcgccattgagcgtcgctgcaaacactacgtggaacttctcgtggcccagggagctgatgtccacgcccaggcccgtgggcgcttcttccagcccaag
I A I E R R C K H Y V E L L V A Q G A D V H A Q A R G R F F Q P K
gatgaggggggctacttctactttggggagctgcccctgtcgctggctgcctgcaccaaccagccccacattgtcaactacctgacggagaacccccac
D E G G Y F Y F G E L P L S L A A C T N Q P H I V N Y L T E N P H
aagaaggcggacatgcggcgccaggactcgcgaggcaacacagtgctgcatgcgctggtggccattgctgacaacacccgtgagaacaccaagtttgtt
K K A D M R R Q D S R G N T V L H A L V A I A D N T R E N T K F V
accaagatgtacgacctgctgctgctcaagtgtgcccgcctcttccccgacagcaacctggaggccgtgctcaacaacgacggcctctcgcccctcatg
T K M Y D L L L L K C A R L F P D S N L E A V L N N D G L S P L M
atggctgccaagacgggcaagattgggatctttcagcacatcatccggcgggaggtgacggatgaggcggccgctcatcatcaccatcatcattga
207
Chapter 6. Appendix
M A A K T G K I G I F Q H I I R R E V T D E A A A H H H H H H -
hsDDX3X_aa122-582
acc atg aaa cat cac cat cac cat cac ccc atg
M K H H H H H H P M
agc gat tac gac atc ccc act act gag aat ctt tat ttt cag ggc ggc aac agc cgc tgg
S D Y D I P T T E N L Y F Q G G N S R W
tgc gat aaa agc gat gag gat gac tgg agc aag ccg ctg ccg ccg agc gaa cgc ctg gag
C D K S D E D D W S K P L P P S E R L E
caa gaa ctg ttt agc ggt ggt aac acc ggt att aac ttt gaa aag tac gac gat atc ccg
Q E L F S G G N T G I N F E K Y D D I P
gtg gag gcg acc ggt aac aac tgc ccg ccg cac att gaa agc ttc agc gac gtt gag atg
V E A T G N N C P P H I E S F S D V E M
ggt gaa atc att atg ggc aac atc gag ctg acc cgt tat acc cgt ccg acc ccg gtg cag
G E I I M G N I E L T R Y T R P T P V Q
aag cat gcg att ccg atc att aag gaa aaa cgt gac ctg atg gcg tgc gcg cag acc ggt
K H A I P I I K E K R D L M A C A Q T G
agc ggt aaa acc gcg gcg ttt ctg ctg ccg atc ctg agc caa att tat agc gat ggt ccg
S G K T A A F L L P I L S Q I Y S D G P
ggt gaa gcg ctg cgt gcg atg aag gaa aac ggt cgt tac ggc cgt cgt aaa cag tat ccg
G E A L R A M K E N G R Y G R R K Q Y P
atc agc ctg gtg ctg gcg ccg acc cgt gag ctg gcg gtt caa att tac gag gaa gcg cgt
I S L V L A P T R E L A V Q I Y E E A R
aaa ttc agc tat cgt agc cgt gtg cgt ccg tgc gtg gtt tac ggt ggc gcg gac atc ggt
K F S Y R S R V R P C V V Y G G A D I G
cag caa att cgt gat ctg gaa cgt ggc tgc cac ctg ctg gtt gcg acc ccg ggt cgt ctg
Q Q I R D L E R G C H L L V A T P G R L
gtt gac atg atg gag cgt ggc aag atc ggc ctg gat ttc tgc aaa tat ctg gtt ctg gac
V D M M E R G K I G L D F C K Y L V L D
gaa gcg gat cgt atg ctg gac atg ggc ttt gag ccg cag atc cgt cgt att gtg gaa caa
E A D R M L D M G F E P Q I R R I V E Q
gat acc atg ccg cca aag ggt gtt cgt cac acc atg atg ttc agc gcg acc ttt ccg aaa
D T M P P K G V R H T M M F S A T F P K
gag atc cag atg ctg gcg cgt gac ttc ctg gat gaa tac att ttt ctg gcg gtg ggt cgt
E I Q M L A R D F L D E Y I F L A V G R
gtt ggc agc acc agc gag aac atc acc caa aag gtg gtt tgg gtg gag gaa agc gac aaa
V G S T S E N I T Q K V V W V E E S D K
cgt agc ttt ctg ctg gat ctg ctg aac gcg acc ggc aag gac agc ctg acc ctg gtg ttc
R S F L L D L L N A T G K D S L T L V F
gtt gaa acc aag aaa ggt gct gac agc ctg gag gat ttt ctg tac cac gaa ggt tat gcg
V E T K K G A D S L E D F L Y H E G Y A
tgc acc agc atc cac ggc gac cgt agc cag cgt gat cgt gag gaa gcg ctg cac caa ttc
C T S I H G D R S Q R D R E E A L H Q F
cgt agc ggc aag agc ccg att ctg gtg gcg acc gcg gtt gcg gcg cgt ggt ctg gat atc
R S G K S P I L V A T A V A A R G L D I
agc aac gtg aaa cac gtt att aac ttt gac ctg ccg agc gat atc gag gaa tat gtg cac
208
Chapter 6. Appendix
S N V K H V I N F D L P S D I E E Y V H
cgt att ggt cgt acc ggc cgt gtt ggt aac ctg ggc ctg gcg acc agc ttc ttt aac gag
R I G R T G R V G N L G L A T S F F N E
cgt aac atc aac att acc aaa gac ctg ctg gat ctg ctg gtt gaa gcg aag caa gag gtg
R N I N I T K D L L D L L V E A K Q E V
ccg agc tgg ctg gag aat atg gcg tat gag cat cac tac aag ggc tga
P S W L E N M A Y E H H Y K G -
hsDDX3X
atg gcc gga aaa cct atc cct aac cct ctg ctg ggg ctg gac tca acc gaa ctg
M A G K P I P N P L L G L D S T E L
att acc tct ctg tat aag aag gct gga act atg tcc cac gtg gca gtg gag aac gca ctg
I T S L Y K K A G T M S H V A V E N A L
ggc ctg gac cag cag ttc gca ggc ctg gac ctg aac agc tcc gat aat cag tct gga ggc
G L D Q Q F A G L D L N S S D N Q S G G
agc acc gcc tcc aag ggc agg tac atc ccc cct cac ctg cgg aat aga gag gcc aca aag
S T A S K G R Y I P P H L R N R E A T K
ggc ttc tat gac aag gat tct agc ggc tgg tcc tct agc aag gac aag gat gcc tac tcc
G F Y D K D S S G W S S S K D K D A Y S
tct ttt ggc tct agg agc gac tcc cgc ggc aag agc tcc ttc ttt tcc gat agg ggc tct
S F G S R S D S R G K S S F F S D R G S
ggc agc agg ggc cgc ttt gac gat agg ggc aga agc gac tat gat ggc atc ggc tcc aga
G S R G R F D D R G R S D Y D G I G S R
ggc gac agg tct ggc ttc ggc aag ttt gag agg gga gga aac agc agg tgg tgc gac aag
G D R S G F G K F E R G G N S R W C D K
tcc gat gag gac gat tgg tct aag cca ctg cca cca agc gag cgg ctg gag cag gag ctg
S D E D D W S K P L P P S E R L E Q E L
ttc agc gga ggc aac acc ggc atc aat ttt gag aag tac gac gat atc ccc gtg gag gcc
F S G G N T G I N F E K Y D D I P V E A
aca ggc aac aat tgt cct cca cac atc gag tcc ttc tct gat gtg gag atg ggc gag atc
T G N N C P P H I E S F S D V E M G E I
atc atg ggc aac atc gag ctg acc cgc tat aca cgg cca acc ccc gtg cag aag cac gcc
I M G N I E L T R Y T R P T P V Q K H A
atc cct atc atc aag gag aag cgg gac ctg atg gca tgc gca cag aca ggc tcc ggc aag
I P I I K E K R D L M A C A Q T G S G K
acc gca gcc ttt ctg ctg ccc atc ctg agc cag atc tac tcc gat gga cct gga gag gcc
T A A F L L P I L S Q I Y S D G P G E A
ctg agg gca atg aag gag aat ggc cgc tac ggc cgg aga aag cag tat cct atc agc ctg
L R A M K E N G R Y G R R K Q Y P I S L
gtg ctg gcc cca acc agg gag ctg gcc gtg cag atc tac gag gag gcc cgc aag ttc tct
V L A P T R E L A V Q I Y E E A R K F S
tat agg agc cgc gtg cgg cct tgc gtg gtg tac gga gga gca gac atc gga cag cag atc
Y R S R V R P C V V Y G G A D I G Q Q I
cgg gat ctg gag aga ggc tgt cac ctg ctg gtg gca acc cca ggc cgg ctg gtg gac atg
R D L E R G C H L L V A T P G R L V D M
atg gag aga ggc aag atc ggc ctg gat ttc tgt aag tat ctg gtg ctg gac gag gcc gat
209
Chapter 6. Appendix
M E R G K I G L D F C K Y L V L D E A D
cgg atg ctg gac atg ggc ttt gag ccc cag atc agg cgc atc gtg gag cag gat aca atg
R M L D M G F E P Q I R R I V E Q D T M
ccc cct aag ggc gtg aga cac aca atg atg ttc agc gcc acc ttt cct aag gag atc cag
P P K G V R H T M M F S A T F P K E I Q
atg ctg gcc cgg gac ttc ctg gat gag tac atc ttt ctg gcc gtg ggc aga gtg ggc agc
M L A R D F L D E Y I F L A V G R V G S
aca tcc gag aac atc acc cag aag gtg gtg tgg gtg gag gag tct gac aag cgg agc ttt
T S E N I T Q K V V W V E E S D K R S F
ctg ctg gat ctg ctg aat gcc aca ggc aag gac tcc ctg acc ctg gtg ttc gtg gag aca
L L D L L N A T G K D S L T L V F V E T
aag aag ggc gcc gac tct ctg gag gat ttt ctg tac cac gag ggc tat gca tgc acc tcc
K K G A D S L E D F L Y H E G Y A C T S
atc cac ggc gac cgg agc cag aga gat agg gag gag gcc ctg cac cag ttc cgc tcc ggc
I H G D R S Q R D R E E A L H Q F R S G
aag tct cca atc ctg gtg gca aca gca gtg gca gca agg ggc ctg gat atc tct aac gtg
K S P I L V A T A V A A R G L D I S N V
aag cac gtg atc aat ttt gac ctg ccc agc gat atc gag gag tat gtg cac aga atc gga
K H V I N F D L P S D I E E Y V H R I G
agg acc gga agg gtg gga aac ctg ggc ctg gcc aca tcc ttc ttt aac gag aga aac atc
R T G R V G N L G L A T S F F N E R N I
aac atc acc aag gac ctg ctg gat ctg ctg gtg gag gcc aag cag gag gtg ccc agc tgg
N I T K D L L D L L V E A K Q E V P S W
ctg gag aat atg gcc tac gag cac cac tat aag ggc tct agc agg ggc cgg agc aag tcc
L E N M A Y E H H Y K G S S R G R S K S
tct agg ttc tcc gga gga ttt gga gca agg gac tac aga cag agc tcc gga gcc tct agc
S R F S G G F G A R D Y R Q S S G A S S
tcc tct ttc agc tcc tct cgg gcc agc tcc tct aga agc ggc ggc ggc ggc cac ggc agc
S S F S S S R A S S S R S G G G G H G S
tcc cgc ggc ttc ggc ggc ggc ggc tat ggc ggc ttt tac aac tca gac ggc tac gga ggg
S R G F G G G G Y G G F Y N S D G Y G G
aac tac aac tca cag ggc gtg gac tgg tgg gga aac tag
N Y N S Q G V D W W G N -
eGFP
atgagcaaaggcgaagaactgtttaccggcgtggtgccgattctggtggaactggatggcgatgtgaacggccataaatttagcgtgagcggcgaaggc
M S K G E E L F T G V V P I L V E L D G D V N G H K F S V S G E G
gaaggcgatgcgacctatggcaaactgaccctgaaatttatttgcaccaccggcaaactgccggtgccgtggccgaccctggtgaccacctttagctat
E G D A T Y G K L T L K F I C T T G K L P V P W P T L V T T F S Y
ggcgtgcagtgctttagccgctatccggatcatatgaaacagcatgatttttttaaaagcgcgatgccggaaggctatgtgcaggaacgcaccattttt
G V Q C F S R Y P D H M K Q H D F F K S A M P E G Y V Q E R T I F
tttaaagatgatggcaactataaaacccgcgcggaagtgaaatttgaaggcgataccctggtgaaccgcattgaactgaaaggcattgattttaaagaa
F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I D F K E
gatggcaacattctgggccataaactggaatataactataacagccataacgtgtatattatggcggataaacagaaaaacggcattaaagtgaacttt
D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K N G I K V N F
aaaattcgccataacattgaagatggcagcgtgcagctggcggatcattatcagcagaacaccccgattggcgatggcccggtgctgctgccggataac
210
Chapter 6. Appendix
K I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N
cattatctgagcacccagagcgcgctgagcaaagatccgaacgaaaaacgcgatcatatggtgctgctggaatttgtgaccgcggcgggcattacccat
H Y L S T Q S A L S K D P N E K R D H M V L L E F V T A A G I T H
ggcatggatgaactgtataaa
G M D E L Y K
hsTRPV4-cGFP
atg gcg gat tcc agc gaa ggc ccc cgc gcg ggg ccc ggg gag gtg gct gag ctc ccc ggg
M A D S S E G P R A G P G E V A E L P G
gat gag agt ggc acc cca ggt ggg gag gct ttt cct ctc tcc tcc ctg gcc aat ctg ttt
D E S G T P G G E A F P L S S L A N L F
gag ggg gag gat ggc tcc ctt tcg ccc tca ccg gct gat gcc agt cgc cct gct ggc cca
E G E D G S L S P S P A D A S R P A G P
ggc gat ggg cga cca aat ctg cgc atg aag ttc cag ggc gcc ttc cgc aag ggg gtg ccc
G D G R P N L R M K F Q G A F R K G V P
aac ccc atc gat ctg ctg gag tcc acc cta tat gag tcc tcg gtg gtg cct ggg ccc aag
N P I D L L E S T L Y E S S V V P G P K
aaa gca ccc atg gac tca ctg ttt gac tac ggc acc tat cgt cac cac tcc agt gac aac
K A P M D S L F D Y G T Y R H H S S D N
aag agg tgg agg aag aag atc ata gag aag cag ccg cag agc ccc aaa gcc cct gcc cct
K R W R K K I I E K Q P Q S P K A P A P
cag ccg ccc ccc atc ctc aaa gtc ttc aac cgg cct atc ctc ttt gac atc gtg tcc cgg
Q P P P I L K V F N R P I L F D I V S R
ggc tcc act gct gac ctg gac ggg ctg ctc cca ttc ttg ctg acc cac aag aaa cgc cta
G S T A D L D G L L P F L L T H K K R L
act gat gag gag ttt cga gag cca tct acg ggg aag acc tgc ctg ccc aag gcc ttg ctg
T D E E F R E P S T G K T C L P K A L L
aac ctg agc aat ggc cgc aac gac acc atc cct gtg ctg ctg gac atc gcg gag cgc acc
N L S N G R N D T I P V L L D I A E R T
ggc aac atg agg gag ttc att aac tcg ccc ttc cgt gac atc tac tat cga ggt cag aca
G N M R E F I N S P F R D I Y Y R G Q T
gcc ctg cac atc gcc att gag cgt cgc tgc aaa cac tac gtg gaa ctt ctc gtg gcc cag
A L H I A I E R R C K H Y V E L L V A Q
gga gct gat gtc cac gcc cag gcc cgt ggg cgc ttc ttc cag ccc aag gat gag ggg ggc
G A D V H A Q A R G R F F Q P K D E G G
tac ttc tac ttt ggg gag ctg ccc ctg tcg ctg gct gcc tgc acc aac cag ccc cac att
Y F Y F G E L P L S L A A C T N Q P H I
gtc aac tac ctg acg gag aac ccc cac aag aaa gcc gac atg agg agg cag gac agc agg
V N Y L T E N P H K K A D M R R Q D S R
ggc aac acc gtg ctg cac gcc ctg gtg gcc atc gcc gac aac acc agg gag aac acc aag
G N T V L H A L V A I A D N T R E N T K
ttc gtg acc aag atg tac gac ctg ctg ctg ctg aag tgc gcc agg ctg ttc ccc gac agc
F V T K M Y D L L L L K C A R L F P D S
aac ctg gag gcc gtg ctg aac aac gac ggc ctg agc ccc ctg atg atg gcc gcc aag acc
N L E A V L N N D G L S P L M M A A K T
ggc aag att ggg atc ttt cag cac atc atc cgg cgg gag gtg acg gat gag gac aca cgg
211
Chapter 6. Appendix
G K I G I F Q H I I R R E V T D E D T R
cac ctg tcc cgc aag ttc aag gac tgg gcc tat ggg cca gtg tat tcc tcg ctt tat gac
H L S R K F K D W A Y G P V Y S S L Y D
ctc tcc tcc ctg gac acg tgt ggg gaa gag gcc tcc gtg ctg gag atc ctg gtg tac aac
L S S L D T C G E E A S V L E I L V Y N
agc aag att gag aac cgc cac gag atg ctg gct gtg gag ccc atc aat gaa ctg ctg cgg
S K I E N R H E M L A V E P I N E L L R
gac aag tgg cgc aag ttc ggg gcc gtc tcc ttc tac atc aac gtg gtc tcc tac ctg tgt
D K W R K F G A V S F Y I N V V S Y L C
gcc atg gtc atc ttc act ctc acc gcc tac tac cag ccg ctg gag ggc aca ccg ccg tac
A M V I F T L T A Y Y Q P L E G T P P Y
cct tac cgc acc acg gtg gac tac ctg cgg ctg gct ggc gag gtc att acg ctc ttc act
P Y R T T V D Y L R L A G E V I T L F T
ggg gtc ctg ttc ttc ttc acc aac atc aaa gac ttg ttc atg aag aaa tgc cct gga gtg
G V L F F F T N I K D L F M K K C P G V
aat tct ctc ttc att gat ggc tcc ttc cag ctg ctc tac ttc atc tac tct gtc ctg gtg
N S L F I D G S F Q L L Y F I Y S V L V
atc gtc tca gca gcc ctc tac ctg gca ggg atc gag gcc tac ctg gcc gtg atg gtc ttt
I V S A A L Y L A G I E A Y L A V M V F
gcc ctg gtc ctg ggc tgg atg aat gcc ctt tac ttc acc cgt ggg ctg aag ctg acg ggg
A L V L G W M N A L Y F T R G L K L T G
acc tat agc atc atg atc cag aag att ctc ttc aag gac ctt ttc cga ttc ctg ctc gtc
T Y S I M I Q K I L F K D L F R F L L V
tac ttg ctc ttc atg atc ggc tac gct tca gcc ctg gtc tcc ctc ctg aac ccg tgt gcc
Y L L F M I G Y A S A L V S L L N P C A
aac atg aag gtg tgc aat gag gac cag acc aac tgc aca gtg ccc act tac ccc tcg tgc
N M K V C N E D Q T N C T V P T Y P S C
cgt gac agc gag acc ttc agc acc ttc ctc ctg gac ctg ttt aag ctg acc atc ggc atg
R D S E T F S T F L L D L F K L T I G M
ggc gac ctg gag atg ctg agc agc acc aag tac ccc gtg gtc ttc atc atc ctg ctg gtg
G D L E M L S S T K Y P V V F I I L L V
acc tac atc atc ctc acc ttt gtg ctg ctc ctc aac atg ctc att gcc ctc atg ggc gag
T Y I I L T F V L L L N M L I A L M G E
aca gtg ggc cag gtc tcc aag gag agc aag cac atc tgg aag ctg cag tgg gcc acc acc
T V G Q V S K E S K H I W K L Q W A T T
atc ctg gac att gag cgc tcc ttc ccc gta ttc ctg agg aag gcc ttc cgc tct ggg gag
I L D I E R S F P V F L R K A F R S G E
atg gtc acc gtg ggc aag agc tcg gac ggc act cct gac cgc agg tgg tgc ttc agg gtg
M V T V G K S S D G T P D R R W C F R V
gat gag gtg aac tgg tct cac tgg aac cag aac ttg ggc atc atc aac gag gac ccg ggc
D E V N W S H W N Q N L G I I N E D P G
aag aat gag acc tac cag tat tat ggc ttc tcg cat acc gtg ggc cgc ctc cgc agg gat
K N E T Y Q Y Y G F S H T V G R L R R D
cgc tgg tcc tcg gtg gta ccc cgc gtg gtg gaa ctg aac aag aac tcg aac ccg gac gag
R W S S V V P R V V E L N K N S N P D E
gtg gtg gtg cct ctg gac agc atg ggg aac ccc cgc tgc gat ggc cac cag cag ggt tac
V V V P L D S M G N P R C D G H Q Q G Y
ccc cgc aag tgg agg act gat gac gcc ccg ctc atg gtg tgc aag tat gag gag ctg ttc
212
Chapter 6. Appendix
P R K W R T D D A P L M V C K Y E E L F
acc ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc
T G V V P I L V E L D G D V N G H K F S
gtg tcc ggc gag agc gag ggc gat gcc acg tac ggc aag ctg acc atg aag ttc atc tgc
V S G E S E G D A T Y G K L T M K F I C
acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acg tac ggc gtg
T T G K L P V P W P T L V T T L T Y G V
cag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg
Q C F S R Y P D H M K Q H D F F K S A M
ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gat gac ggc aac tac aag acc
P E G Y V Q E R T I F F K D D G N Y K T
cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc atc
R A E V K F E G D T L V N R I E L K G I
gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac aac tac aac agc cac
D F K E D G N I L G H K L E Y N Y N S H
aac gtc tat atc atg gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag atc cgc
N V Y I M A D K Q K N G I K V N F K I R
cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc
H N I E D G S V Q L A D H Y Q Q N T P I
ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc aag ctg agc
G D G P V L L P D N H Y L S T Q S K L S
aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg
K D P N E K R D H M V L L E F V T A A G
atc act ctc ggc atg gac gag ctg tac aag tag
I T L G M D E L Y K -
hsTRPV4 R232C-cGFP
see hsTRPV4-cGFP, c.694C>T, p. Arg232C
hsTRPV4 R269C-cGFP
see hsTRPV4-cGFP, c.805C>T, p. Arg269C
hsTRPV4-cFLAG
atg gcg gat tcc agc gaa ggc ccc cgc gcg ggg ccc
M A D S S E G P R A G P
ggg gag gtg gct gag ctc ccc ggg gat gag agt ggc acc cca ggt ggg gag gct ttt cct
G E V A E L P G D E S G T P G G E A F P
ctc tcc tcc ctg gcc aat ctg ttt gag ggg gag gat ggc tcc ctt tcg ccc tca ccg gct
L S S L A N L F E G E D G S L S P S P A
gat gcc agt cgc cct gct ggc cca ggc gat ggg cga cca aat ctg cgc atg aag ttc cag
D A S R P A G P G D G R P N L R M K F Q
213
Chapter 6. Appendix
ggc gcc ttc cgc aag ggg gtg ccc aac ccc atc gat ctg ctg gag tcc acc cta tat gag
G A F R K G V P N P I D L L E S T L Y E
tcc tcg gtg gtg cct ggg ccc aag aaa gca ccc atg gac tca ctg ttt gac tac ggc acc
S S V V P G P K K A P M D S L F D Y G T
tat cgt cac cac tcc agt gac aac aag agg tgg agg aag aag atc ata gag aag cag ccg
Y R H H S S D N K R W R K K I I E K Q P
cag agc ccc aaa gcc cct gcc cct cag ccg ccc ccc atc ctc aaa gtc ttc aac cgg cct
Q S P K A P A P Q P P P I L K V F N R P
atc ctc ttt gac atc gtg tcc cgg ggc tcc act gct gac ctg gac ggg ctg ctc cca ttc
I L F D I V S R G S T A D L D G L L P F
ttg ctg acc cac aag aaa cgc cta act gat gag gag ttt cga gag cca tct acg ggg aag
L L T H K K R L T D E E F R E P S T G K
acc tgc ctg ccc aag gcc ttg ctg aac ctg agc aat ggc cgc aac gac acc atc cct gtg
T C L P K A L L N L S N G R N D T I P V
ctg ctg gac atc gcg gag cgc acc ggc aac atg agg gag ttc att aac tcg ccc ttc cgt
L L D I A E R T G N M R E F I N S P F R
gac atc tac tat cga ggt cag aca gcc ctg cac atc gcc att gag cgt cgc tgc aaa cac
D I Y Y R G Q T A L H I A I E R R C K H
tac gtg gaa ctt ctc gtg gcc cag gga gct gat gtc cac gcc cag gcc cgt ggg cgc ttc
Y V E L L V A Q G A D V H A Q A R G R F
ttc cag ccc aag gat gag ggg ggc tac ttc tac ttt ggg gag ctg ccc ctg tcg ctg gct
F Q P K D E G G Y F Y F G E L P L S L A
gcc tgc acc aac cag ccc cac att gtc aac tac ctg acg gag aac ccc cac aag aaa
A C T N Q P H I V N Y L T E N P H K K
gcc gac atg agg agg cag gac agc agg ggc aac acc gtg ctg cac gcc ctg gtg gcc
A D M R R Q D S R G N T V L H A L V A
atc gcc gac aac acc agg gag aac acc aag
I A D N T R E N T K
ttc gtg acc aag atg tac gac ctg ctg ctg ctg aag tgc gcc agg ctg ttc ccc gac agc
F V T K M Y D L L L L K C A R L F P D S
aac ctg gag gcc gtg ctg aac aac gac ggc ctg agc ccc ctg atg atg gcc gcc aag acc
N L E A V L N N D G L S P L M M A A K T
ggc aag att ggg atc ttt cag cac atc atc cgg cgg gag
G K I G I F Q H I I R R E
gtg acg gat gag gac aca cgg cac ctg tcc cgc aag ttc aag gac tgg gcc tat ggg cca
V T D E D T R H L S R K F K D W A Y G P
gtg tat tcc tcg ctt tat gac ctc tcc tcc ctg gac acg tgt ggg gaa gag gcc tcc gtg
V Y S S L Y D L S S L D T C G E E A S V
ctg gag atc ctg gtg tac aac agc aag att gag aac cgc cac gag atg ctg gct gtg gag
L E I L V Y N S K I E N R H E M L A V E
ccc atc aat gaa ctg ctg cgg gac aag tgg cgc aag ttc ggg gcc gtc tcc ttc tac atc
P I N E L L R D K W R K F G A V S F Y I
aac gtg gtc tcc tac ctg tgt gcc atg gtc atc ttc act ctc acc gcc tac tac cag ccg
N V V S Y L C A M V I F T L T A Y Y Q P
ctg gag ggc aca ccg ccg tac cct tac cgc acc acg gtg gac tac ctg cgg ctg gct ggc
L E G T P P Y P Y R T T V D Y L R L A G
gag gtc att acg ctc ttc act ggg gtc ctg ttc ttc ttc acc aac atc aaa gac ttg ttc
E V I T L F T G V L F F F T N I K D L F
214
Chapter 6. Appendix
atg aag aaa tgc cct gga gtg aat tct ctc ttc att gat ggc tcc ttc cag ctg ctc tac
M K K C P G V N S L F I D G S F Q L L Y
ttc atc tac tct gtc ctg gtg atc gtc tca gca gcc ctc tac ctg gca ggg atc gag gcc
F I Y S V L V I V S A A L Y L A G I E A
tac ctg gcc gtg atg gtc ttt gcc ctg gtc ctg ggc tgg atg aat gcc ctt tac ttc acc
Y L A V M V F A L V L G W M N A L Y F T
cgt ggg ctg aag ctg acg ggg acc tat agc atc atg atc cag aag att ctc ttc aag gac
R G L K L T G T Y S I M I Q K I L F K D
ctt ttc cga ttc ctg ctc gtc tac ttg ctc ttc atg atc ggc tac gct tca gcc ctg gtc
L F R F L L V Y L L F M I G Y A S A L V
tcc ctc ctg aac ccg tgt gcc aac atg aag gtg tgc aat gag gac cag acc aac tgc aca
S L L N P C A N M K V C N E D Q T N C T
gtg ccc act tac ccc tcg tgc cgt gac agc gag acc ttc agc acc ttc ctc ctg gac ctg
V P T Y P S C R D S E T F S T F L L D L
ttt aag ctg acc atc ggc atg ggc gac ctg gag atg ctg agc agc acc aag tac ccc
F K L T I G M G D L E M L S S T K Y P
gtg gtc ttc atc atc ctg ctg gtg acc tac atc atc ctc acc ttt gtg
V V F I I L L V T Y I I L T F V
ctg ctc ctc aac atg ctc att gcc ctc atg ggc gag aca gtg ggc cag gtc tcc aag gag
L L L N M L I A L M G E T V G Q V S K E
agc aag cac atc tgg aag ctg cag tgg gcc acc acc atc ctg gac att gag cgc tcc ttc
S K H I W K L Q W A T T I L D I E R S F
ccc gta ttc ctg agg aag gcc ttc cgc tct ggg gag atg gtc acc gtg ggc aag agc tcg
P V F L R K A F R S G E M V T V G K S S
gac ggc act cct gac cgc agg tgg tgc ttc agg gtg gat gag gtg aac tgg tct cac tgg
D G T P D R R W C F R V D E V N W S H W
aac cag aac ttg ggc atc atc aac gag gac ccg ggc aag aat gag acc tac cag tat tat
N Q N L G I I N E D P G K N E T Y Q Y Y
ggc ttc tcg cat acc gtg ggc cgc ctc cgc agg gat cgc tgg tcc tcg gtg gta ccc cgc
G F S H T V G R L R R D R W S S V V P R
gtg gtg gaa ctg aac aag aac tcg aac ccg gac gag gtg gtg gtg cct ctg gac agc atg
V V E L N K N S N P D E V V V P L D S M
ggg aac ccc cgc tgc gat ggc cac cag cag ggt tac ccc cgc aag tgg agg act gat gac
G N P R C D G H Q Q G Y P R K W R T D D
gcc ccg ctc aat tcg tcg aca agc ttc tcg agc atg cat cta gat gac tat gat gac
A P L G N L N S S T S F S S M H L D D
tat aaa gac gat gac gac tag
Y K D D D K -
hsTRPV4 R232C-cFLAG
see hsTRPV4-cFLAG, c.694C>T, p. Arg232C
215
Chapter 6. Appendix
hsTRPV4 R269C-cFLAG
see hsTRPV4-cFLAG, c.805C>T, p. Arg269C
hsPACSIN1-nV5
atggcaggcaagccaatccctaaccctctgctgggcctggacagcaccgaattgatcacaagtttgtacaaaaaagcaggcacc
M A G K P I P N P L L G L D S T E L I T S L Y K K A G T
atg agc agc agc tac gac gag gcc agc ctg gcc ccc gag gag acc acc gac agc ttc tgg
M S S S Y D E A S L A P E E T T D S F W
gag gtg ggc aac tac aag agg acc gtg aag agg atc gac gac ggc cac agg ctg tgc aac
E V G N Y K R T V K R I D D G H R L C N
gac ctg atg aac tgc gtg cag gag agg gcc aag atc gag aag gcc tac ggc cag cag ctg
D L M N C V Q E R A K I E K A Y G Q Q L
acc gac tgg gcc aag agg tgg agg cag ctg atc gag aag ggc ccc cag tac ggc agc ctg
T D W A K R W R Q L I E K G P Q Y G S L
gag agg gcc tgg ggc gcc atc atg acc gag gcc gac aag gtg agc gag ctg cac cag gag
E R A W G A I M T E A D K V S E L H Q E
gtg aag aac aac ctg ctg aac gag gac ctg gag aag gtg aag aac tgg cag aag gac gcc
V K N N L L N E D L E K V K N W Q K D A
tac cac aag cag atc atg ggc ggc ttc aag gag acc aag gag gcc gag gac ggc ttc agg
Y H K Q I M G G F K E T K E A E D G F R
aag gcc cag aag ccc tgg gcc aag aag atg aag gag ctg gag gcc gcc aag aag gcc tac
K A Q K P W A K K M K E L E A A K K A Y
cac ctg gcc tgc aag gag gag aag ctg gcc atg acc agg gag atg aac agc aag acc gag
H L A C K E E K L A M T R E M N S K T E
cag agc gtg acc ccc gag cag cag aag aag ctg cag gac aag gtg gac aag tgc aag cag
Q S V T P E Q Q K K L Q D K V D K C K Q
gac gtg cag aag acc cag gag aag tac gag aag gtg ctg gag gac gtg ggc aag acc acc
D V Q K T Q E K Y E K V L E D V G K T T
ccc cag tac atg gag aac atg gag cag gtg ttc gag cag tgc cag cag ttc gag gag aag
P Q Y M E N M E Q V F E Q C Q Q F E E K
agg ctg gtg ttc ctg aag gag gtg ctg ctg gac atc aag agg cac ctg aac ctg gcc gag
R L V F L K E V L L D I K R H L N L A E
aac agc agc tac atc cac gtg tac agg gag ctg gag cag gcc atc agg ggc gcc gac gcc
N S S Y I H V Y R E L E Q A I R G A D A
cag gag gac ctg agg tgg ttc agg agc acc agc ggc ccc ggc atg ccc atg aac tgg ccc
Q E D L R W F R S T S G P G M P M N W P
cag ttc gag gag tgg aac ccc gac ctg ccc cac acc acc acc aag aag gag aag cag ccc
Q F E E W N P D L P H T T T K K E K Q P
aag aag gcc gag ggc gtg gcc ctg acc aac gcc acc ggc gcc gtg gag agc acc agc cag
K K A E G V A L T N A T G A V E S T S Q
gcc ggc gac agg ggc agc gtg agc agc tac gac agg ggc cag ccc tac gcc acc gag tgg
A G D R G S V S S Y D R G Q P Y A T E W
agc gac gac gag agc ggc aac ccc ttc ggc ggc agc gag acc aac ggc ggc gcc aac ccc
S D D E S G N P F G G S E T N G G A N P
ttc gag gac gac agc aag ggc gtg agg gtg agg gcc ctg tac gac tac gac ggc cag gag
216
Chapter 6. Appendix
F E D D S K G V R V R A L Y D Y D G Q E
cag gac gag ctg agc ttc aag gcc ggc gac gag ctg acc aag ctg ggc gag gag gac gag
Q D E L S F K A G D E L T K L G E E D E
cag ggc tgg tgc agg ggc agg ctg gac agc ggc cag ctg ggc ctg tac ccc gcc aac tac
Q G W C R G R L D S G Q L G L Y P A N Y
gtg gag gcc atc tag
V E A I -
hsPACSIN2-nV5
atggcaggcaagccaatccctaaccctctgctgggcctggacagcaccgaattgatcacaagtttgtacaaaaaagcaggc
M A G K P I P N P L L G L D S T E L I T S L Y K K A G
atg agc gtg acc tac gac gac agc gtg ggc gtg gag gtg agc agc gac agc ttc tgg gag
M S V T Y D D S V G V E V S S D S F W E
gtg ggc aac tac aag agg acc gtg aag agg atc gac gac ggc cac agg ctg tgc agc gac
V G N Y K R T V K R I D D G H R L C S D
ctg atg aac tgc ctg cac gag agg gcc agg atc gag aag gcc tac gcc cag cag ctg acc
L M N C L H E R A R I E K A Y A Q Q L T
gag tgg gcc agg agg tgg agg cag ctg gtg gag aag ggc ccc cag tac ggc acc gtg gag
E W A R R W R Q L V E K G P Q Y G T V E
aag gcc tgg atg gcc ttc atg agc gag gcc gag agg gtg agc gag ctg cac ctg gag gtg
K A W M A F M S E A E R V S E L H L E V
aag gcc agc ctg atg aac gac gac ttc gag aag atc aag aac tgg cag aag gag gcc ttc
K A S L M N D D F E K I K N W Q K E A F
cac aag cag atg atg ggc ggc ttc aag gag acc aag gag gcc gag gac ggc ttc agg aag
H K Q M M G G F K E T K E A E D G F R K
gcc cag aag ccc tgg gcc aag aag ctg aag gag gtg gag gcc gcc aag aag gcc cac cac
A Q K P W A K K L K E V E A A K K A H H
gcc gcc tgc aag gag gag aag ctg gcc atc agc agg gag gcc aac agc aag gcc gac ccc
A A C K E E K L A I S R E A N S K A D P
agc ctg aac ccc gag cag ctg aag aag ctg cag gac aag atc gag aag tgc aag cag gac
S L N P E Q L K K L Q D K I E K C K Q D
gtg ctg aag acc aag gag aag tac gag aag agc ctg aag gag ctg gac cag ggc acc ccc
V L K T K E K Y E K S L K E L D Q G T P
cag tac atg gag aac atg gag cag gtg ttc gag cag tgc cag cag ttc gag gag aag agg
Q Y M E N M E Q V F E Q C Q Q F E E K R
ctg agg ttc ttc agg gag gtg ctg ctg gag gtg cag aag cac ctg gac ctg agc aac gtg
L R F F R E V L L E V Q K H L D L S N V
gcc ggc tac aag gcc atc tac cac gac ctg gag cag agc atc agg gcc gcc gac gcc gtg
A G Y K A I Y H D L E Q S I R A A D A V
gag gac ctg agg tgg ttc agg gcc aac cac ggc ccc ggc atg gcc atg aac tgg ccc cag
E D L R W F R A N H G P G M A M N W P Q
ttc gag gag tgg agc gcc gac ctg aac agg acc ctg agc agg agg gag aag aag aag gcc
F E E W S A D L N R T L S R R E K K K A
acc gac ggc gtg acc ctg acc ggc atc aac cag acc ggc gac cag agc ctg ccc agc aag
T D G V T L T G I N Q T G D Q S L P S K
ccc agc agc acc ctg aac gtg ccc agc aac ccc gcc cag agc gcc cag agc cag agc agc
217
Chapter 6. Appendix
P S S T L N V P S N P A Q S A Q S Q S S
tac aac ccc ttc gag gac gag gac gac acc ggc agc acc gtg agc gag aag gac gac acc
Y N P F E D E D D T G S T V S E K D D T
aag gcc aag aac gtg agc agc tac gag aag acc cag agc tac ccc acc gac tgg agc gac
K A K N V S S Y E K T Q S Y P T D W S D
gac gag agc aac aac ccc ttc agc agc acc gac gcc aac ggc gac agc aac ccc ttc gac
D E S N N P F S S T D A N G D S N P F D
gac gac gcc acc agc ggc acc gag gtg agg gtg agg gcc ctg tac gac tac gag ggc cag
D D A T S G T E V R V R A L Y D Y E G Q
gag cac gac gag ctg agc ttc aag gcc ggc gac gag ctg acc aag atg gag gac gag gac
E H D E L S F K A G D E L T K M E D E D
gag cag ggc tgg tgc aag ggc agg ctg gac aac ggc cag gtg ggc ctg tac ccc gcc aac
E Q G W C K G R L D N G Q V G L Y P A N
tac gtg gag gcc atc cag tag
Y V E A I Q -
hsPACSIN3-nV5
atggcaggcaagccaatccctaaccctctgctgggcctggacagcaccgaattgatcacaagtttgtacaaaaaagcaggc
M A G K P I P N P L L G L D S T E L I T S L Y K K A G
atg gcc ccc gag gag gac gcc ggc ggc gag gcc ctg ggc ggc agc ttc tgg gag gcc ggc
M A P E E D A G G E A L G G S F W E A G
aac tac agg agg acc gtg cag agg gtg gag gac ggc cac agg ctg tgc ggc gac ctg gtg
N Y R R T V Q R V E D G H R L C G D L V
agc tgc ttc cag gag agg gcc agg atc gag aag gcc tac gcc cag cag ctg gcc gac tgg
S C F Q E R A R I E K A Y A Q Q L A D W
gcc agg aag tgg agg ggc acc gtg gag aag ggc ccc cag tac ggc acc ctg gag aag gcc
A R K W R G T V E K G P Q Y G T L E K A
tgg cac gcc ttc ttc acc gcc gcc gag agg ctg agc gcc ctg cac ctg gag gtg agg gag
W H A F F T A A E R L S A L H L E V R E
aag ctg cag ggc cag gac agc gag agg gtg agg gcc tgg cag agg ggc gcc ttc cac agg
K L Q G Q D S E R V R A W Q R G A F H R
ccc gtg ctg ggc ggc ttc agg gag agc agg gcc gcc gag gac ggc ttc agg aag gcc cag
P V L G G F R E S R A A E D G F R K A Q
aag ccc tgg ctg aag agg ctg aag gag gtg gag gcc agc aag aag agc tac cac gcc gcc
K P W L K R L K E V E A S K K S Y H A A
agg aag gac gag aag acc gcc cag acc agg gag agc cac gcc aag gcc gac agc gcc gtg
R K D E K T A Q T R E S H A K A D S A V
agc cag gag cag ctg agg aag ctg cag gag agg gtg gag agg tgc gcc aag gag gcc gag
S Q E Q L R K L Q E R V E R C A K E A E
aag acc aag gcc cag tac gag cag acc ctg gcc gag ctg cac agg tac acc ccc agg tac
K T K A Q Y E Q T L A E L H R Y T P R Y
atg gag gac atg gag cag gcc ttc gag acc tgc cag gcc gcc gag agg cag agg ctg ctg
M E D M E Q A F E T C Q A A E R Q R L L
ttc ttc aag gac atg ctg ctg acc ctg cac cag cac ctg gac ctg agc agc agc gag aag
F F K D M L L T L H Q H L D L S S S E K
ttc cac gag ctg cac agg gac ctg cac cag ggc atc gag gcc gcc agc gac gag gag gac
218
Chapter 6. Appendix
F H E L H R D L H Q G I E A A S D E E D
ctg agg tgg tgg agg agc acc cac ggc ccc ggc atg gcc atg aac tgg ccc cag ttc gag
L R W W R S T H G P G M A M N W P Q F E
gag tgg agc ctg gac acc cag agg acc atc agc agg aag gag aag ggc ggc agg agc ccc
E W S L D T Q R T I S R K E K G G R S P
gac gag gtg acc ctg acc agc atc gtg ccc acc agg gac ggc acc gcc ccc ccc ccc cag
D E V T L T S I V P T R D G T A P P P Q
agc ccc ggc agc ccc ggc acc ggc cag gac gag gag tgg agc gac gag gag agc ccc agg
S P G S P G T G Q D E E W S D E E S P R
aag gcc gcc acc ggc gtg agg gtg agg gcc ctg tac gac tac gcc ggc cag gag gcc gac
K A A T G V R V R A L Y D Y A G Q E A D
gag ctg agc ttc agg gcc ggc gag gag ctg ctg aag atg agc gag gag gac gag cag ggc
E L S F R A G E E L L K M S E E D E Q G
tgg tgc cag ggc cag ctg cag agc ggc agg atc ggc ctg tac ccc gcc aac tac gtg gag
W C Q G Q L Q S G R I G L Y P A N Y V E
tgc gtg ggc gcc tag
C V G A -
PACSIN1+2-nV5
attt aag tgt cat tgg cag att acc acc atg gca ggc aag cca atc cct aac cct ctg ctg
F K C H W Q I T T M A G K P I P N P L L
ggc ctg gac agc acc gaa ttg atc aca agt ttg tac aaa aaa gca ggc acc atg tcc agc
G L D S T E L I T S L Y K K A G T M S S
tcc tac gat gag gcc tca ctg gta gaa gtg tcc agc gac agc ttc tgg gag gtc ggg aac
S Y D E A S L V E V S S D S F W E V G N
tac aag cgg act gtg aag cgg atc gac gat ggc cac cgc ctg tgc agc gac ctc atg aac
Y K R T V K R I D D G H R L C S D L M N
tgc ctg cat gag cgg gcg cgc atc gag aag gcg tat gcg cag cag ctc act gag tgg gcc
C L H E R A R I E K A Y A Q Q L T E W A
cgg cgc tgg agg cag ctc gtg gag aaa ggg ccc cag tac ggg acc gtg gag aag gcc tgg
R R W R Q L V E K G P Q Y G T V E K A W
atg gcc ttc atg tcc gag gca gag agg gtg agc gag ctg cac ctc gag gtg aag gcc tca
M A F M S E A E R V S E L H L E V K A S
ctg atg aac gat gac ttc gag aag atc aag aac tgg cag aag gaa gcc ttt cac aag cag
L M N D D F E K I K N W Q K E A F H K Q
atg atg ggc ggc ttc aag gag acc aag gaa gct gag gac ggc ttt cgg aag gca cag aag
M M G G F K E T K E A E D G F R K A Q K
ccc tgg gcc aag aag ctg aaa gag gta gaa gca gca aag aaa gcc cac cat gca gcg tgc
P W A K K L K E V E A A K K A H H A A C
aaa gag gag aag ctg gct atc tca cga gaa gcc aac agc aag gca gac cca tcc ctc aac
K E E K L A I S R E A N S K A D P S L N
cct gaa cag ctc aag aaa ttg caa gac aaa ata gaa aag tgc aag caa gat gtt ctt aag
P E Q L K K L Q D K I E K C K Q D V L K
acc aaa gag aag tat gag aag tcc ctg aaa gaa ctc gac cag ggc aca ccc cag tac atg
T K E K Y E K S L K E L D Q G T P Q Y M
gag aac atg gag cag tgt ttg agc agt gcc agc agt tcg agg aga aaa cgc ctt cgc ttc
219
Chapter 6. Appendix
E N M E Q C L S S A S S S R R K R L R F
ttc cgg gag gtt ctg ctg gag gtt cag aag cac cta gac ctg tcc aat gtg gct ggc tac
F R E V L L E V Q K H L D L S N V A G Y
aaa gcc att tac cat gac ctg gag cag agc atc aga gca gct gat gca gtg gag gac ctc
K A I Y H D L E Q S I R A A D A V E D L
aga tgg ttc cgc agc acc agt ggc ccc ggc atg ccc atg aac tgg ccc cag ttt gag gag
R W F R S T S G P G M P M N W P Q F E E
tgg aac cca gac ctt cct cac acc acc acc aag aag gag aaa cag cct aag aag gca gag
W N P D L P H T T T K K E K Q P K K A E
gga gtg gcg ctg acc aat gcc act ggg gcg gta gag tcc aca tcc cag gct ggg gac cgc
G V A L T N A T G A V E S T S Q A G D R
ggc agt gtt agc agc tac gac aga ggc cag ccc tac gcc acc gag tgg tca gac gac gag
G S V S S Y D R G Q P Y A T E W S D D E
agt ggg aac ccc ttt ggg ggc agt gag acc aac ggg ggc gcc aac ccc ttt gag gac gac
S G N P F G G S E T N G G A N P F E D D
tcc aag gga gtg cgc gtg cgg gca ctc tac gac tat gac ggc cag gag cag gac gag ctc
S K G V R V R A L Y D Y D G Q E Q D E L
agc ttt aag gcc gga gac gaa ctc acc aag ctg ggc gag gag gat gag cag ggc tgg tgc
S F K A G D E L T K L G E E D E Q G W C
cgt ggg cgg ctg gac agc ggg cag ctg ggc ctc tac cct gcc aac tac gtg gag gct atc
R G R L D S G Q L G L Y P A N Y V E A I
tag aac cca gct
- N P A
PACSIN1+3-nV5
attt aag tgt cat tgg cag att acc acc atg gca ggc aag cca atc cct aac cct ctg ctg
F K C H W Q I T T M A G K P I P N P L L
ggc ctg gac agc acc gaa ttg atc aca agt ttg tac aaa aaa gca ggc acc atg tcc agc
G L D S T E L I T S L Y K K A G T M S S
tcc tac gat gag gcc tca ctg atg gcc ccc gag gag gac gcc ggc ggc gag gcc ctg ggc
S Y D E A S L M A P E E D A G G E A L G
ggc agc ttc tgg gag gcc ggc aac tac agg agg acc gtg cag agg gtg gag gac ggc cac
G S F W E A G N Y R R T V Q R V E D G H
agg ctg tgc ggc gac ctg gtg agc tgc ttc cag gag agg gcc agg atc gag aag gcc tac
R L C G D L V S C F Q E R A R I E K A Y
gcc cag cag ctg gcc gac tgg gcc agg aag tgg agg ggc acc gtg gag aag ggc ccc cag
A Q Q L A D W A R K W R G T V E K G P Q
tac ggc acc ctg gag aag gcc tgg cac gcc ttc ttc acc gcc gcc gag agg ctg agc gcc
Y G T L E K A W H A F F T A A E R L S A
ctg cac ctg gag gtg agg gag aag ctg cag ggc cag gac agc gag agg gtg agg gcc tgg
L H L E V R E K L Q G Q D S E R V R A W
cag agg ggc gcc ttc cac agg ccc gtg ctg ggc ggc ttc agg gag agc agg gcc gcc gag
Q R G A F H R P V L G G F R E S R A A E
gac ggc ttc agg aag gcc cag aag ccc tgg ctg aag agg ctg aag gag gtg gag gcc agc
D G F R K A Q K P W L K R L K E V E A S
aag aag agc tac cac gcc gcc agg aag gac gag aag acc gcc cag acc agg gag agc cac
220
Chapter 6. Appendix
K K S Y H A A R K D E K T A Q T R E S H
gcc aag gcc gac agc gcc gtg agc cag gag cag ctg agg aag ctg cag gag agg gtg gag
A K A D S A V S Q E Q L R K L Q E R V E
agg tgc gcc aag gag gcc gag aag acc aag gcc cag tac gag cag acc ctg gcc gag ctg
R C A K E A E K T K A Q Y E Q T L A E L
cac agg tac acc ccc agg tac atg gag gac atg gag cag gcc ttc gag acc tgc cag gcc
H R Y T P R Y M E D M E Q A F E T C Q A
gcc gag agg cag agg ctg ctg ttc ttc aag gac atg ctg ctg acc ctg cac cag cac ctg
A E R Q R L L F F K D M L L T L H Q H L
gac ctg agc agc agc gag aag ttc cac gag ctg cac agg gac ctg cac cag ggc atc gag
D L S S S E K F H E L H R D L H Q G I E
gcc gcc agc gac gag gag gac ctc aga tgg ttc cgc agc acc agt ggc ccc ggc atg ccc
A A S D E E D L R W F R S T S G P G M P
atg aac tgg ccc cag ttt gag gag tgg aac cca gac ctt cct cac acc acc acc aag aag
M N W P Q F E E W N P D L P H T T T K K
gag aaa cag cct aag aag gca gag gga gtg gcg ctg acc aat gcc act ggg gcg gta gag
E K Q P K K A E G V A L T N A T G A V E
tcc aca tcc cag gct ggg gac cgc ggc agt gtt agc agc tac gac aga ggc cag ccc tac
S T S Q A G D R G S V S S Y D R G Q P Y
gcc acc gag tgg tca gac gac gag agt ggg aac ccc ttt ggg ggc agt gag acc aac ggg
A T E W S D D E S G N P F G G S E T N G
ggc gcc aac ccc ttt gag gac gac tcc aag gga gtg cgc gtg cgg gca ctc tac gac tat
G A N P F E D D S K G V R V R A L Y D Y
gac ggc cag gag cag gac gag ctc agc ttt aag gcc gga gac gaa ctc acc aag ctg ggc
D G Q E Q D E L S F K A G D E L T K L G
gag gag gat gag cag ggc tgg tgc cgt ggg cgg ctg gac agc ggg cag ctg ggc ctc tac
E E D E Q G W C R G R L D S G Q L G L Y
cct gcc aac tac gtg gag gct atc tag aac cca gct
P A N Y V E A I - N P A
PACSIN3+2-nV5
atg gca ggc aag cca atc cct aac cct ctg ctg ggc ctg gac agc acc gaa ttg atc aca
M A G K P I P N P L L G L D S T E L I T
agt ttg tac aaa aaa gca ggc tcc acc gta gaa gtg tcc agc gac agc ttc tgg gag gtc
S L Y K K A G S T V E V S S D S F W E V
ggg aac tac aag cgg act gtg aag cgg atc gac gat ggc cac cgc ctg tgc agc gac ctc
G N Y K R T V K R I D D G H R L C S D L
atg aac tgc ctg cat gag cgg gcg cgc atc gag aag gcg tat gcg cag cag ctc act gag
M N C L H E R A R I E K A Y A Q Q L T E
tgg gcc cgg cgc tgg agg cag ctc gtg gag aaa ggg ccc cag tac ggg acc gtg gag aag
W A R R W R Q L V E K G P Q Y G T V E K
gcc tgg atg gcc ttc atg tcc gag gca gag agg gtg agc gag ctg cac ctc gag gtg aag
A W M A F M S E A E R V S E L H L E V K
gcc tca ctg atg aac gat gac ttc gag aag atc aag aac tgg cag aag gaa gcc ttt cac
A S L M N D D F E K I K N W Q K E A F H
221
Chapter 6. Appendix
aag cag atg atg ggc ggc ttc aag gag acc aag gaa gct gag gac ggc ttt cgg aag gca
K Q M M G G F K E T K E A E D G F R K A
cag aag ccc tgg gcc aag aag ctg aaa gag gta gaa gca gca aag aaa gcc cac cat gca
Q K P W A K K L K E V E A A K K A H H A
gcg tgc aaa gag gag aag ctg gct atc tca cga gaa gcc aac agc aag gca gac cca tcc
A C K E E K L A I S R E A N S K A D P S
ctc aac cct gaa cag ctc aag aaa ttg caa gac aaa ata gaa aag tgc aag caa gat gtt
L N P E Q L K K L Q D K I E K C K Q D V
ctt aag acc aaa gag aag tat gag aag tcc ctg aaa gaa ctc gac cag ggc aca ccc cag
L K T K E K Y E K S L K E L D Q G T P Q
tac atg gag aac atg gag cag tgt ttg agc agt gcc agc agt tcg agg aga aaa cgc ctt
Y M E N M E Q C L S S A S S S R R K R L
cgc ttc ttc cgg gag gtt ctg ctg gag gtt cag aag cac cta gac ctg tcc aat gtg gct
R F F R E V L L E V Q K H L D L S N V A
ggc tac aaa gcc att tac cat gac ctg gag cag agc atc aga gca gct gat gca gtg gag
G Y K A I Y H D L E Q S I R A A D A V E
gac ctg cgc tgg tgg cgc agc acc cac ggg cca ggc atg gcc atg aac tgg cca cag ttc
D L R W W R S T H G P G M A M N W P Q F
gag gag tgg tcc ttg gac aca cag agg aca atc agc cgg aaa gag aag ggt ggc cgg agc
E E W S L D T Q R T I S R K E K G G R S
cct gat gag gtt acc ctg acc agc att gtg cct aca aga gat ggc acc gca ccc cca ccc
P D E V T L T S I V P T R D G T A P P P
cag tcc ccg ggg tcc cca ggc acg ggg cag gat gag gag tgg tca gat gaa gag agt ccc
Q S P G S P G T G Q D E E W S D E E S P
cgg aag gct gcc acc ggg gtt cgg gtg agg gca ctc tat gac tac gct ggc cag gaa gct
R K A A T G V R V R A L Y D Y A G Q E A
gat gag ctg agc ttc cga gca ggg gag gag ctg ctg aag atg agt gag gag gac gag cag
D E L S F R A G E E L L K M S E E D E Q
ggc tgg tgc caa ggc cag ttg cag agt ggc cgc att ggc ctg tac cct gcc aac tac gtg
G W C Q G Q L Q S G R I G L Y P A N Y V
gag tgt gtg ggc gcc tag
E C V G A -
hsTRPML1-cGFP
atgaccgcgccggcgggcccgcgcggcagcgaaaccgaacgcctgctgaccccgaacccgggctatggcacccaggcgggcccgagcccggcgccgccg
M T A P A G P R G S E T E R L L T P N P G Y G T Q A G P S P A P P
accccgccggaagaagaagatctgcgccgccgcctgaaatatttttttatgagcccgtgcgataaatttcgcgcgaaaggccgcaaaccgtgcaaactg
T P P E E E D L R R R L K Y F F M S P C D K F R A K G R K P C K L
atgctgcaggtggtgaaaattctggtggtgaccgtgcagctgattctgtttggcctgagcaaccagctggcggtgacctttcgcgaagaaaacaccatt
M L Q V V K I L V V T V Q L I L F G L S N Q L A V T F R E E N T I
gcgtttcgccatctgtttctgctgggctatagcgatggcgcggatgatacctttgcggcgtatacccgcgaacagctgtatcaggcgatttttcatgcg
A F R H L F L L G Y S D G A D D T F A A Y T R E Q L Y Q A I F H A
gtggatcagtatctggcgctgccggatgtgagcctgggccgctatgcgtatgtgcgcggcggcggcgatccgtggaccaacggcagcggcctggcgctg
V D Q Y L A L P D V S L G R Y A Y V R G G G D P W T N G S G L A L
tgccagcgctattatcatcgcggccatgtggatccggcgaacgatacctttgatattgatccgatggtggtgaccgattgcattcaggtggatccgccg
C Q R Y Y H R G H V D P A N D T F D I D P M V V T D C I Q V D P P
222
Chapter 6. Appendix
gaacgcccgccgccgccgccgagcgatgatctgaccctgctggaaagcagcagcagctataaaaacctgaccctgaaatttcataaactggtgaacgtg
E R P P P P P S D D L T L L E S S S S Y K N L T L K F H K L V N V
accattcattttcgcctgaaaaccattaacctgcagagcctgattaacaacgaaattccggattgctatacctttagcgtgctgattacctttgataac
T I H F R L K T I N L Q S L I N N E I P D C Y T F S V L I T F D N
aaagcgcatagcggccgcattccgattagcctggaaacccaggcgcatattcaggaatgcaaacatccgagcgtgtttcagcatggcgataacagcttt
K A H S G R I P I S L E T Q A H I Q E C K H P S V F Q H G D N S F
cgcctgctgtttgatgtggtggtgattctgacctgcagcctgagctttctgctgtgcgcgcgcagcctgctgcgcggctttctgctgcagaacgaattt
R L L F D V V V I L T C S L S F L L C A R S L L R G F L L Q N E F
gtgggctttatgtggcgccagcgcggccgcgtgattagcctgtgggaacgcctggaatttgtgaacggctggtatattctgctggtgaccagcgatgtg
V G F M W R Q R G R V I S L W E R L E F V N G W Y I L L V T S D V
ctgaccattagcggcaccattatgaaaattggcattgaagcgaaaaacctggcgagctatgatgtgtgcagcattctgctgggcaccagcaccctgctg
L T I S G T I M K I G I E A K N L A S Y D V C S I L L G T S T L L
gtgtgggtgggcgtgattcgctatctgaccttttttcataactataacattctgattgcgaccctgcgcgtggcgctgccgagcgtgatgcgcttttgc
V W V G V I R Y L T F F H N Y N I L I A T L R V A L P S V M R F C
tgctgcgtggcggtgatttatctgggctattgcttttgcggctggattgtgctgggcccgtatcatgtgaaatttcgcagcctgagcatggtgagcgaa
C C V A V I Y L G Y C F C G W I V L G P Y H V K F R S L S M V S E
tgcctgtttagcctgattaacggcgatgatatgtttgtgacctttgcggcgatgcaggcgcagcagggccgcagcagcctggtgtggctgtttagccag
C L F S L I N G D D M F V T F A A M Q A Q Q G R S S L V W L F S Q
ctgtatctgtatagctttattagcctgtttatttatatggtgctgagcctgtttattgcgctgattaccggcgcgtatgataccattaaacatccgggc
L Y L Y S F I S L F I Y M V L S L F I A L I T G A Y D T I K H P G
ggcgcgggcgcggaagaaagcgaactgcaggcgtatattgcgcagtgccaggatagcccgaccagcggcaaatttcgccgcggcagcggcagcgcgtgc
G A G A E E S E L Q A Y I A Q C Q D S P T S G K F R R G S G S A C
agcctgctgtgctgctgcggccgcgatccgagcgaagaacatagcctgctggtgaacatgagcaaaggcgaagaactgtttaccggcgtggtgccgatt
S L L C C C G R D P S E E H S L L V N M S K G E E L F T G V V P I
ctggtggaactggatggcgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggcgatgcgacctatggcaaactgaccctgaaatttatt
L V E L D G D V N G H K F S V S G E G E G D A T Y G K L T L K F I
tgcaccaccggcaaactgccggtgccgtggccgaccctggtgaccacctttagctatggcgtgcagtgctttagccgctatccggatcatatgaaacag
C T T G K L P V P W P T L V T T F S Y G V Q C F S R Y P D H M K Q
catgatttttttaaaagcgcgatgccggaaggctatgtgcaggaacgcaccattttttttaaagatgatggcaactataaaacccgcgcggaagtgaaa
H D F F K S A M P E G Y V Q E R T I F F K D D G N Y K T R A E V K
tttgaaggcgataccctggtgaaccgcattgaactgaaaggcattgattttaaagaagatggcaacattctgggccataaactggaatataactataac
F E G D T L V N R I E L K G I D F K E D G N I L G H K L E Y N Y N
agccataacgtgtatattatggcggataaacagaaaaacggcattaaagtgaactttaaaattcgccataacattgaagatggcagcgtgcagctggcg
S H N V Y I M A D K Q K N G I K V N F K I R H N I E D G S V Q L A
gatcattatcagcagaacaccccgattggcgatggcccggtgctgctgccggataaccattatctgagcacccagagcgcgctgagcaaagatccgaac
D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q S A L S K D P N
gaaaaacgcgatcatatggtgctgctggaatttgtgaccgcggcgggcattacccatggcatggatgaactgtataaatag
E K R D H M V L L E F V T A A G I T H G M D E L Y K -
YAP1_WW1 domain-His6
cat atg agc agc ttt gag atc ccg gac gac gtg ccg ctg ccg gcg ggt tgg gag atg gcg
H M S S F E I P D D V P L P A G W E M A
aag acc agc agc ggt cag cgt tac ttt ctg aac cac atc gac cag acc acc acc tgg caa
K T S S G Q R Y F L N H I D Q T T T W Q
gat ccg cgt aag gcg atg gag aat ctg tat ttt caa ggt cat cat cat cat cat cat taa
D P R K A M E N L Y F Q G H H H H H H -
223
Chapter 6. Appendix
gga tcc
G S
YAP1_WW2 domain-His6
cat atg agc gcg agc ggt ccg ctg ccg gat ggt tgg gag cag gcg atg acc caa gat ggt
H M S A S G P L P D G W E Q A M T Q D G
gaa atc tac tac atc aac cac aag aac aag acc acc agc tgg ctg gac ccg cgt ctg gac
E I Y Y I N H K N K T T S W L D P R L D
ccg cgt gag aat ctg tat ttt caa ggt cat cat cat cat cat cat taa
P R E N L Y F Q G H H H H H H -
hsYAP1_WW1+WW2 domains-His6
cat atg agc agc ttt gag atc ccg gac gac gtg ccg ctg ccg gcg ggt tgg gag atg gcg
H M S S F E I P D D V P L P A G W E M A
aag acc agc agc ggt cag cgt tac ttt ctg aac cac atc gac cag acc acc acc tgg caa
K T S S G Q R Y F L N H I D Q T T T W Q
gat ccg cgt aag gcg atg ctg agc cag atg aac gtg acc gcg ccg acc agc ccg ccg gtt
D P R K A M L S Q M N V T A P T S P P V
cag caa aac atg atg aac agc gcg agc ggt ccg ctg ccg gat ggt tgg gag cag gcg atg
Q Q N M M N S A S G P L P D G W E Q A M
acc caa gat ggt gaa atc tac tac atc aac cac aag aac aag acc acc agc tgg ctg gac
T Q D G E I Y Y I N H K N K T T S W L D
ccg cgt ctg gac ccg cgt gag aat ctg tat ttt caa ggt cat cat cat cat cat cat taa
P R L D P R E N L Y F Q G H H H H H H -
224
Chapter 7. List of Figures
7 List of Figures
1.1 Multiple alignment phylogenetic tree of the mammalian TRP channel superfamily. . . . . . . . . . . . . . 17
1.2 Structures of human TRP channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.3 TRP channel PPI network extracted from the TRIP Database. . . . . . . . . . . . . . . . . . . . . 20
1.4 Distribution of TRPV4 channelopathy mutations . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.1 Purification of in vivo biotinylated and structural intact TRPV4 N-terminal constructs . . . . . . . . . . . . 85
4.2 Outline of the proteomics workflow performed in this work . . . . . . . . . . . . . . . . . . . . . . 88
4.3 UDMSE measurements revealed up to 64 probable hsV4N protein interactors. . . . . . . . . . . . . . . 89
4.4 Interaction map and GO Enrichment Components and Processes of hsV4N interactome proteins detected via UDMSE 90
4.5 Interaction map and GO Enrichment Components and Processes of hsV4N interactome proteins detected via
UDMSE with Ca2+ sample supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.7 Overlap of proteins detected in respective detected in respective UDMSE experiments without (A) and with Ca2+
sample supplementation (B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.6 Interaction map and GO Enrichment Components and Processes of hsV4 ARD wt Avi, hsV4 ARD R232C Avi and
hsV4 ARD K276E Avi interactome proteins detected via UDMSE without and with Ca2+ sample supplementation 95
4.8 Purification of recombinant TRPV4 N-terminal constructs and human DDX3X . . . . . . . . . . . . . . 100
4.9 hsV4 ARD, but not hsV4N, decreases the DDX3X ATPase activity . . . . . . . . . . . . . . . . . . . 102
4.10 XL-MS confirms a direct interaction between hsV4N and DDX3X . . . . . . . . . . . . . . . . . . . 103
4.11 hsV4 ARD interacts with dxRNA-bound DDX3X . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.12 dsRNA binding changes the tryptophan environment in DDX3X dramatically. . . . . . . . . . . . . . . . 106
4.13 Inhibition of DDX3X ATPase activity is enhanced in the presence of neuropathy-causing TRPV4 R232C mutation. 107
4.14 Co-Immunoprecipitations of various full-length human TRPV4-GFP constructs and DDX3X-V5 in HEK293 cells show
an increased DDX3X-V5 pulldown with the neuropathy-causing TRPV4-R232C mutation . . . . . . . . . . 108
4.15 Immunofluorescence images of HEK293T cells co-transfected with TRPV4-GFP (green) and DDX3X-V5 (magenta)
reveal an cytosolic DDX3X densificaton, especially in the presence of TRPV4-R232C-GFP. . . . . . . . . . 109
4.16 Human PACSIN1 and 3, but not PACSIN2, dampen human TRPV4-mediated Ca2+-influx in transiently transfected
HEK293T cells upon hypotonicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.17 Human PACSIN1 does not dampen human TRPV4-mediated Ca2+-influx in HEK293T cells transiently transfected
with TRPV4 R269C upon hypotonicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.18 PACSIN chimeras hint towards regulation modes between PACSIN1 and PACSIN3 on TRPV4-mediated Ca-influx in
tar HEK293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.19 Purification of hsV4 ARD wt and 15N-RhoA 15N-RhoA . . . . . . . . . . . . . . . . . . . . . . . 120
4.20 NMR experiments revealed an decreased PPI between 15N-RhoA and the neuropathy-causing mutation R269C in
hsV4 ARD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.21 Relative signal intensity changes of RhoA in the presence of unlabeled hsV4 ARD wt and R269C, respectively. . 123
4.22 Interaction sites of hsV4 ARD with 15N-RhoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.23 Purification of recombinant TRPV4 N-terminal constructs and human ITCH . . . . . . . . . . . . . . . 126
4.24 ITCH and hsV4N are direct interaction partners . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4.25 In vitro ubiquitination assay shows ubiquitination of hsV4N by ITCH . . . . . . . . . . . . . . . . . . 130
225
Chapter 7. List of Figures
4.26 Cross-link mass spectrometry (XL-MS) measurements showing inter- and/or intramolecular interactions between
ITCH molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
4.27 Fluorescence microscopy images reveal stress fiber formation upon TRPV4 activation in stably transfected HEK293
cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4.28 ELSEXY GFP TRITC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.1 Tissue specificity of neuropathy-causing TRPV4 mutations occur due to aberrant tissue specific protein-protein in-
teractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.1 TRP channel PPI network extracted from the TRIP Database. . . . . . . . . . . . . . . . . . . . . 143
6.2 Purification of unbiotinylated hsV4 ARD Avi constructs . . . . . . . . . . . . . . . . . . . . . . . 152
6.3 hsV4 ARD does not influence or inhibit the assay components lactate dehydrogenase and pyruvate kinase . . 177
6.4 Overlay of spectra of 1H-15N RhoA 2D HSCQs of 15N-RhoA on its own and with hsV4 ARD wt . . . . . . . 183
6.5 Overlay of spectra of 1H-15N RhoA 2D HSCQs of 15N-RhoA on its own and with hsV4 R269C . . . . . . . 184
6.6 Annotated samples in BN PAGE, submitted to MS measurements . . . . . . . . . . . . . . . . . . . 189
6.7 Fluorescence microscopy images show no effect of GSK-101 or HC-067 on the cytoskeleton of unstransfected
HEK293 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
6.8 Pearson Correlation Coefficients (PCC, r) of respective fluorescence marker pair. . . . . . . . . . . . . . 193
6.9 M-values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
6.10 Fluorescence microscopy images of HEK293 cells stabily transfected with hsTRPV4-cGFP . . . . . . . . . 201
6.11 Stably transfected HEK293 cells express funtional hsTRPV4-cGFP. . . . . . . . . . . . . . . . . . . 201
6.12 Fluorescence microscopy images of HEK293 cells stabily transfected with hsTRPML1-cGFP . . . . . . . . 202
6.13 Purification of recombinant human ITCH WW domains . . . . . . . . . . . . . . . . . . . . . . . 202
6.14 Purification of recombinant human YAP1 WW domains . . . . . . . . . . . . . . . . . . . . . . . 203
226
Chapter 8. List of Tables
8 List of Tables
2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 General buffer and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3 Buffer and solutions for purification of hsV4N, hsV4N∆122 and hsV4N∆132 constructs. hsV4N∆132 is also re-
ferred to as hsV4 ARD, hsV4N∆122 as hsV4 ARD-PRR. . . . . . . . . . . . . . . . . . . . . . . 34
2.4 Buffer and solutions for purification of hsRhoA . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.5 Buffer and solutions for purification of hsDDX3X_aa122-582 (DDX3X) . . . . . . . . . . . . . . . . . 36
2.6 Buffer and solutions for purification of hsITCH . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.7 Buffer and solutions for purification of hsITCH WW domain constructs . . . . . . . . . . . . . . . . . 38
2.8 Buffer and solutions for purification of TRPC 3, 4 and 6 ARDs . . . . . . . . . . . . . . . . . . . . 39
2.9 Buffer and solutions for purification of hsYAP-WW domains . . . . . . . . . . . . . . . . . . . . . 40
2.10 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.11 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.12 Oligonucleotides for Quickchange PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.12 Oligonucleotides for Quickchange PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.13 Oligonucleotides for Gibbson Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.13 Oligonucleotides for Gibbson Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.14 Sequencing oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.15 Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD,
hsV4N∆122 as hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the
Hellmich workgroup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.15 Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD,
hsV4N∆122 as hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the
Hellmich workgroup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.15 Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD,
hsV4N∆122 as hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the
Hellmich workgroup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.15 Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD,
hsV4N∆122 as hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the
Hellmich workgroup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.15 Expression plasmids generated and used in the course of this work. hsV4N∆132 is also referred as hsV4 ARD,
hsV4N∆122 as hsV4 ARD-PRR. The nomenclature of the plasmids refers to the employed nomenclature at the
Hellmich workgroup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.16 Characteristics of proteins and peptides used in this thesis. . . . . . . . . . . . . . . . . . . . . . 52
2.17 Characteristics of proteins and peptides used in this thesis. . . . . . . . . . . . . . . . . . . . . . 53
2.18 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.19 Media and supplements for cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.20 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.20 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.21 Laboratory equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
227
Chapter 8. List of Tables
2.22 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.1 DpnI digestion reaction mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.2 Reaction mixtures for Gibson Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.3 Composition of the stacking and separation gel for SDS-PAGE . . . . . . . . . . . . . . . . . . . . 61
3.3 Composition of the stacking and separation gel for SDS-PAGE . . . . . . . . . . . . . . . . . . . . 62
3.4 Used primary antibody solutions and blocking solutions . . . . . . . . . . . . . . . . . . . . . . . 62
3.5 Used secondary antibody solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.6 Used SEC columns, manufactured by GE Healthcare . . . . . . . . . . . . . . . . . . . . . . . . 64
3.7 Used plasmid DNA amounts for transfections of HEK293T cells with Lipofectamine ®LTX . . . . . . . . . 65
3.8 Used plasmid amounts for stable transfections of HEK293 cells with Lipofectamine ®LTX . . . . . . . . . 67
3.9 Antibodies used for cell immunostainings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.10 Expression conditions of human N-terminal TRPV4 (hsV4N) constructs . . . . . . . . . . . . . . . . . 70
3.11 Expression conditions of human RhoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.12 Expression conditions of hsDDX3X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.13 Expression conditions of human ITCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.14 Expression conditions of human ITCH WW domains . . . . . . . . . . . . . . . . . . . . . . . . 75
3.15 Expression conditions of human YAP WW domains . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.16 Sample composition for the in vitro-ATPase assay . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.17 Sample composition for the in vitro-Ubiquitinylation assay . . . . . . . . . . . . . . . . . . . . . . 81
4.1 Sample overview of hsV4N interactome pulldowns with HEK293 cytosolic protein extracts . . . . . . . . . 87
6.1 List of human TRP channel protein interactors deposited in the TRIP database . . . . . . . . . . . . . . 144
6.2 List of known TRPV4 interactors deposited in the TRIP database . . . . . . . . . . . . . . . . . . . 146
6.3 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4N protein interactome via UDMSE in
this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
6.4 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4N protein interactome in the presence
of 2 mM Ca2+ via UDMSE in this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
6.5 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD wt protein interactome deter-
mined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
6.6 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD wt protein interactome in the
presence of 2 mM Ca2+ determined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . . 160
6.7 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD R232C protein interactome
determined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.8 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD R232C protein interactome in
the presence of 2 mM Ca2+ determined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . 166
6.9 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD K276E protein interactome
determined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
6.10 List of cytoplasmic RNP granule and stress granule proteins found in the hsV4 ARD K276E protein interactome in
the presence of 2 mM Ca2+ determined via UDMSE in this work . . . . . . . . . . . . . . . . . . . . 173
6.11 MS data of ubiquitinated hsV4N after in vitro ubiquitination assay with ITCH . . . . . . . . . . . . . . . 185
6.12 MS data of BN PAGE complexes of ITCH and hsV4N . . . . . . . . . . . . . . . . . . . . . . . . 190
6.14 P and M-values of GFP (TRPV4-cGFP) and TRITC (F-actin stained with Phalloidon TRITC) images of stably trans-
fected HEK293 cells upon various treatments, determined and calculated by ELSEXY (see also figures 4.28, 6.9
and 6.8). PCC = Pearson correlation coefficient r . . . . . . . . . . . . . . . . . . . . . . . . . 194
6.15 P and M-values of TRITC (F-actin stained with Phalloidon TRITC) and DAPI (Nucleus) images of stably transfected
HEK293 cells upon various treatments, determined and calculated by ELSEXY (see also figures 4.28, 6.9 and 6.8).
PCC = Pearson correlation coefficient r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
228
Chapter 8. List of Tables
6.16 P and M-values of GFP (TRPV4-cGFP) and DAPI (Nucleus) images of stably transfected HEK293 cells upon various
treatments, determined and calculated by ELSEXY (see also figures 4.28, 6.9 and 6.8). PCC = Pearson correlation
coefficient r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
229
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Curriculum Vitae 
Education and Research Experience 
Jan 2019 – Visiting researcher at the research group of Prof. , Johns 
Jun 2019 Hopkins University, USA (Fulbright Fellowship). 
 “The impact of selected TRPV4 interactors upon TRPV4 mediated Ca2+-influx 
 activity” 
 Live Ca2+-influx imaging in cellulo. Immunostaining and subsequent confocal 
 microscopy. Mouse studies on peripheral neuropathies.   
  
Since Apr PhD candidate in chemistry (TransMED as well as Sybille Kalkhof Rose-
2017 Fellowship), Johannes Gutenberg-University (JGU) Mainz 
“Elucidating molecular details in the interactome of the human ion channel 
TRPV4” 
Investigation of direct protein-protein interactions (PPI) via UDMSE, NMR and 
analytical FPLC. Indirect PPI studies via co-IPs and microscopical co-localization 
studies. Developing an in-house software for microscopical co-localization 
analysis. 
Jun 2016 – Master-Thesis, “Investigating the interactome of the TRPV4 N-terminus”,  
Apr 2017 JGU Mainz 
Recombinant expression of human proteins in E. coli, P. pastoris and HEK293T 
cells. Purification via several chromatographic methods, including preparative 
FPLC. Confirming structural protein integrity via CD spectroscopy and 2D-NMR.  
Apr 2015 – Research internship, “AOM-induced colorectal carcinogenesis in PARP-1 k.o. 
Jun 2015 mice”, JGU Mainz 
Inducing colorectal cancer in PARP-1 k.o. mice. Evaluation of carcinogenesis in 
vivo via colonoscopy. Immunohistostaining of colorectal tissues and subsequent 
confocal microscopy. ELISAs for TNF level determination. 
Oct 2014 – Studies in Biomedical Chemistry (M.Sc.), JGU Mainz 
Apr 2017 Elective subjects: Pharmacology, Oncology 
Apr 2014 – Student staff member at the IMG Innovations-Management GmbH 
Mar 2016 Novelty assessment of inventions with focus on industrial property rights in life 
science and chemistry. Composition of technological summaries of approved 
patents in English and German. Acquisition of technology licensees. 
Apr 2014 – Bachelor-Thesis, “Influence of disulfide compounds on the tumor-supressor 
Sep 2014 protein p53”, JGU Mainz 
Cell culture with colorectal cancer cell lines. ELISAs, EMSAs with subsequent 
Western- and Southern blotting. Recombinant expression of human proteins in 
Sf9 insect cells via baculoviruses 
Oct 2013 – Interdisciplinary advanced seminar “Tumor biology, oncological dia-gnostics and 
Apr 2014 novel therapies”, JGU Mainz 
July 2012 Extracurricular research internship, “Expression level of CD91 in leukemic stem 
cells”, JGU Mainz 
Cell culture with suspension cells, FACS 
Oct 2010 – Studies in Biomedical Chemistry (B.Sc.), JGU Mainz 
Sep 2014 Elective subject: Genotoxicology  
Mar 2010 Higher education entrance qualification, “Abitur” 
Gymnasium Kusel 
 
 
 
 
 
 
 
 
Awards  
Nov 2019 Sybille Kalkhof Rose-Stiftung Fellowship  
Sep 2019 Best Talk Award of a young researcher by the association frei für forschung – Neue 
Wege in der Medizin e.V. at the 6th TransMED ScienceDay, JGU Mainz 
Sep 2018 Science Award by the association frei für forschung – Neue Wege in der Medizin 
e.V. at the CRISPR Genome Editing Workshop, JGU Mainz 
Aug 2018 Fulbright Fellowship  
Mar 2018 Best Presentation Award at the 3rd Interdisciplinary symposium JGU Mainz 
Oct 2017 TransMED – Mainz Research School of Translational Biomedicine Fellowship 
Organizing and Teaching Experience 
Oct 2017+ Conference conception and organization of 2nd BioChem Symposium and 3rd Life 
Apr 2018 Science Meeting, JGU Mainz 
since Apr Supervision of 9 undergraduate students during their Bachelor and Master theses 
2017 in (biomedical) chemistry. Conception and lecturing of undergraduate literature 
seminars and practical courses in “Biochemistry 2” 
Oct 2011 – Undergraduate research assistant, JGU Mainz 
Oct 2012 Conception, supervision and correction of exams in the subject “Allgemeine und 
anorganische Chemie I”. 
List of publications 
• McCray BA, Diehl E, Sullivan JM, Aisenberg WH, Zaccor NW, Lau AR, Goretzki B, Hellmich 
UA, Llyod TE, Sumner CJ. „Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA 
interactions and impair actin cytoskeleton regulation“ (Nature Communications 12, 1444 
(2021) 
• Wesenberg LJ, Diehl E, Zähringer TJB, Schollmeyer D, Shimizu A, Yoshida J, Hellmich UA, 
Waldvogel SR. „Twofold Electrochemical C,H-Amination of Activated Benzene 
Derivatives“ (Accepted, Chemistry – A European Journal) 
• Neitzel, C., Seiwert, N., Göder, A., Diehl, E, Weber, C., Nagel, G., Stroh, S., Rasenberger, B., 
Christmann, M., and Fahrer, J. (2019). Lipoic Acid Synergizes with Antineoplastic Drugs in 
Colorectal Cancer by Targeting p53 for Proteasomal Degradation. Cells 8, 794 (2019) 
• Wagner A, Le TA, Brennich M, Klein P, Bader N, Diehl E, Paszek D, Weickhmann AK, Dirdjaja N, 
Krauth-Siegel RL. Inhibitor-Induced Dimerization of an Essential Oxidoreductase from African 
Trypanosomes. Angewandte der Chemie International Edition 58, 3640–3644. (2019) 
• Goretzki B, Glogowski NA, Diehl E, Durchardt-Ferner E, Hacker C, Gaudet R, Hellmich UA. 
Structural Basis of TRPV4 N-Terminus Interaction with Syndapin/PACSIN1-3 and PIP2. Structure 
26, 1583-1593.e5 (2018). 
• Dörsam B, Seiwerth S, Foersch S, Stroh S, Nagel G, Begaliew D, Diehl E, Kraus A, 
McKeague M, Minneker V, Roukos V, Reißig S, Waisman A, Moehler M, Stier A, Mangerich 
A, Dantzer F, Kaina B and Fahrer, J. PARP-1 protects against colorectal tumor induction, but 
promotes inflammation-driven colorectal tumor progression. Proc. Nat. Acad. Sci. 115, 
E4061–E4070 (2018). 
• Wagner A, Diehl E, Krauth-Siegel RL, Hellmich UA. Backbone NMR assignment of 
tryparedoxin, the central protein in the hydroperoxide detoxification cascade of African 
trypanosomes, in the oxidized and reduced form. Biomol NMR Assign. Volume 11, Issue 2, 
pp 193-196 (2017) 
 
Supervised theses 
Jul 2020 –  – Bachelor thesis “A biochemical study of selected TRPV4 
Oct 2020 interaction partners”  
Jun2018 –  Diploma thesis “Investigating the Interactome of the Human Ion Channel 
Mar 2019 TRPV4 and Associated Channelopathies” 
Oct 2017 –  – Bachelor thesis “Biochemische Studien an Ankyrin Repeat 
Jan 2018 Domains von TRPV-Ionenkanälen”  
Conference contributions (Selection) 
Sep 2019 6th TransMed Science Day, JGU Mainz 
“Towards elucidating the channelopathy interactome of the human ion channel 
TRPV4” (talk) 
Sep 2018 Laureate lecture at the CRISPR Genome Editing Workshop, JGU Mainz 
“Why CRISPR? A TR(i)P through the interactome world of the Transient Receptor 
Potential channel 4” 
Mar 2018 3rd Interdisciplinary Symposium, JGU Mainz 
 “Transient receptor potential channels – Building a bridge between Aristotle and 
chili” (talk)