Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.
Author ORCID Identifier
https://orcid.org/0000-0002-0802-5451
AccessType
Campus-Only Access for Five (5) Years
Document Type
dissertation
Degree Name
Doctor of Philosophy (PhD)
Degree Program
Chemistry
Year Degree Awarded
2022
Month Degree Awarded
September
First Advisor
Eric R. Strieter
Subject Categories
Biochemistry
Abstract
Degradation of proteins by the 26S proteasome is a highly regulated process that is mediated by the type of ubiquitin (Ub) modification present on the substrate. Ub can form polyubiquitin chains by modifying itself on any of its seven surface Lys residues and the N-terminal Met. This leads to a multitude of different chain architectures that can decorate substrates. Branched Ub chains, where a single Ub is modified at two different sites, have been shown to be particularly potent signals for degradation. UCH37 is a proteasome associated deubiquitinase (DUB), which is recruited to the proteasome by RPN13. Our lab has shown that the UCH37-RPN13 complex selectively cleaves K48 linked chains when they are part of a branch point, thereby promoting degradation. This thesis addresses how this enzyme selectively binds to K48 linked chains and discriminates between branched and nonbranched ubiquitin. For this we utilized a number of biophysical techniques including HDX-MS, NMR, chemical crosslinking and molecular docking. Our results uncover a novel ubiquitin binding site on the α5-α6 motif of iv UCH37. This site is required for K48 chains binding and debranching. Our results also suggest that two binding poses are possible for a K48 linked chain, however only one pose is likely to enable debranching. Furthermore, using NMR spectroscopy, we also studied the interactions of a K6/K48 branched trimer with UCH37. We have shown that all three subunits of the trimer interact with the enzyme and show unique chemical shifts. We identified a unique set of peaks in the K48-distal subunit of the branched trimer corresponding to a conformation unique to the branched trimer when in complex with UCH37. Our results suggest that stabilization of this unique conformation could be the explanation for branched point selectivity.
DOI
https://doi.org/10.7275/31141906
Recommended Citation
Babik, Sandor, "ELUCIDATING THE INTERACTIONS OF K48-LINKED UBIQUITIN CHAINS WITH UCH37/UCHL5" (2022). Doctoral Dissertations. 2593.
https://doi.org/10.7275/31141906
https://scholarworks.umass.edu/dissertations_2/2593
Creative Commons License
This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License.