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Structural and Functional Studies of Human E3 Ubiquitn Ligase HUWE1

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dc.contributor.advisorSheng, Yi
dc.creatorKhatun, Rahima
dc.date.accessioned2018-05-28T12:52:49Z
dc.date.available2018-05-28T12:52:49Z
dc.date.copyright2017-09-19
dc.date.issued2018-05-28
dc.date.updated2018-05-28T12:52:49Z
dc.degree.disciplineBiology
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractUbiquitin (Ub) acts as an intracellular signal once tagged covalently to the target proteins and regulates a myriad of cellular processes. Ubiquitin is attached to the target protein by the concerted action of E1, E2, and E3 Ub ligase enzymes, in the presence of ATP. During this process, E3 Ub ligases are responsible for the final step of protein ubiquitination and play crucial roles in substrate selectivity and specificity. Therefore, understanding the structure and function of E3 Ub ligases provides valuable mechanistic information regarding their specific roles in Ub-mediated regulatory processes. In this dissertation, we aimed to investigate the structure and function of the human E3 Ub ligase, HUWE1. HUWE1 regulates multiple cellular pathways, including DNA damage response, apoptosis, and transcriptional regulation through controlling the stability and fate of various proteins involved in these pathways. Using multidisciplinary approaches, we studied the functional domains, the regulation, and the new cellular substrates of HUWE1. We report that HUWE1 harbors a previously uncharacterized tandem ubiquitin-binding motif (UBM). It contains three independently folded unique UBMs, and as a tandem, it binds three different Ub chains. Most significantly, this tandem UBM enhances HUWE1-mediated ubiquitination. We also uncovered the mechanism of a X-linked mental retardation mutation, R2981H, of HUWE1. Our results show this mutation disrupts the structure of the UBM that attenuates HUWE1 function. We also investigated the molecular mechanism of the HUWE1 inhibition by ARF. We identified an 8mer ARF peptide that binds HUWE1 and p53 in vivo to inhibit HUWE1-mediated p53 ubiquitination, which results in p53 transcriptional activation. Moreover, this peptide inhibited cancer cell growth in a p53-dependent manner. Finally, we found that HUWE1 directly binds and ubiquitinates -catenin, a master signal transducer in the canonical WNT signaling pathway. Overall, these findings identify a new HUWE1 substrate that provides new knowledge of HUWE1 function in cell regulation and development.
dc.identifier.urihttp://hdl.handle.net/10315/34532
dc.language.isoen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectCellular biology
dc.subject.keywordsMolecular Biology
dc.subject.keywordsBiochemistry
dc.subject.keywordsCell biology
dc.subject.keywordsStructural Biology
dc.subject.keywordsProtein Biochemistry
dc.subject.keywordsBiophysical techniques such as NMR
dc.subject.keywordsCrystallography/ITC/Synergy H4 Plate etc
dc.subject.keywordsGene Cloning
dc.subject.keywordsProtein over expression in Bacteria/mammalian cells
dc.subject.keywordsTissue Culture
dc.subject.keywordsTransfection
dc.subject.keywordsWestern Blotting
dc.subject.keywordsInvitro/In vivo Biochemical analysis
dc.subject.keywordsImaging using specialized Microscopy such as Fluorescence microscopy.
dc.titleStructural and Functional Studies of Human E3 Ubiquitn Ligase HUWE1
dc.typeElectronic Thesis or Dissertation

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