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Autoren: Bluhm, Alina
Titel: Mass spectrometry-based proteomics to study the development of D. melanogaster and telomere-associated proteins with a focus on human ZBTB10
Online-Publikationsdatum: 19-Mär-2019
Erscheinungsdatum: 2019
Sprache des Dokuments: Englisch
Zusammenfassung/Abstract: Mass spectrometry-based proteomics is a versatile tool that can be used to analyze proteins in a global and unbiased fashion. In this thesis, state-of-the-art LC-MS/MS technology has been used to 1) investigate protein expression changes during development by quantitative proteomics, 2) identify novel telomere-associated proteins and 3) determine protein-protein interactions by interactomics. Article I presents the first protein-based study covering the complete life cycle of the commonly used model organism Drosophila melanogaster. Using label-free quantitative mass spectrometry, we generated two large datasets: the lifecycle proteome and a highly time-resolved embryogenesis proteome. We identified stage- and gender-specific proteins, as well as maternally-provided proteins that might play a role in oogenesis or early development. Moreover, we observed only moderate correlation at all time points between mRNA and protein, which is important to consider in developmental studies. By integrating tissue-specific expression data, we further inferred possible roles for uncharacterized proteins in the development of certain tissues. These comprehensive proteomic datasets serve as powerful resources for future investigations. Article II expands our current knowledge about telomere-associated proteins. We identified TTAGGG-repeat binders in 16 vertebrate cell lines by affinity purification coupled to quantitative mass spectrometry. We detected an unexpected loss of telomeric binding by the shelterin complex member TRF1 in non-therian species. In addition, we identified several novel proteins associated with the telomeric sequence presenting interesting candidates for future functional studies at telomeres. In the most recent publication (Article III), we focused on one of these candidates: ZBTB10. We analyzed the binding behavior of ZBTB10 to different telomeric repeats and determined direct binding via its two zinc fingers. While we confirmed binding to the TTAGGG motif, binding affinity for the TTGGGG telomere variant repeat was two-times higher. We observed localization of ZBTB10 at a subset of telomeres in ALT-maintaining U2OS cells. To gain insights into the possible molecular function of ZBTB10, we analyzed the interaction network in two human cell lines by quantitative mass spectrometry. Of note, the shelterin complex members TRF2 and RAP1 were reproducibly identified as ZBTB10 interactors in both cell lines. The N-terminus of TRF2 was defined to be necessary and sufficient for binding ZBTB10. Therefore, ZBTB10 presents a novel telomere-binding protein at ALT telomeres. In conclusion, I investigated a wide range of research questions that can be addressed by MS-based proteomics and interactomics. I then further characterized a novel telomere interactor derived from our phylointeractomics screen.
DDC-Sachgruppe: 570 Biowissenschaften
570 Life sciences
Veröffentlichende Institution: Johannes Gutenberg-Universität Mainz
Organisationseinheit: Externe Einrichtungen
Veröffentlichungsort: Mainz
ROR: https://ror.org/023b0x485
DOI: http://doi.org/10.25358/openscience-3039
URN: urn:nbn:de:hebis:77-diss-1000026902
Version: Original work
Publikationstyp: Dissertation
Nutzungsrechte: Urheberrechtsschutz
Informationen zu den Nutzungsrechten: https://rightsstatements.org/vocab/InC/1.0/
Umfang: getrennte Zählung
Enthalten in den Sammlungen:JGU-Publikationen

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