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|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 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 ARD R269C). In a cooperation with the 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.
|540 Chemistry and allied sciences
|570 Life sciences
|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
|248 Seiten, Illustrationen, Diagramme
|FB 09 Chemie, Pharmazie u. Geowissensch.
|Johannes Gutenberg-Universität Mainz
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