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Author ORCID Identifier

https://orcid.org/0000-0003-0224-9128

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2022

Month Degree Awarded

September

First Advisor

Eric R. Strieter

Second Advisor

D. Joseph Jerry

Third Advisor

Patricia Wadsworth

Fourth Advisor

ChangHui Pak

Subject Categories

Cell Biology | Molecular Biology

Abstract

Cellular protein pools are maintained through the biological processes of synthesis, degradation and quality control. As the dysregulation of these processes has been implicated in diseases such as neurodegeneration and cancer, understanding their functions is critical for drug development. Modification of proteins with ubiquitin may direct them to the proteasome, a large cellular protease complex, for degradation. Yet, the proteasome contains three deubiquitinating enzymes (DUBs) which remove ubiquitin from proteins, potentially altering their fate. As each DUB recognizes specific ubiquitin linkages and architectures, their activity may regulate how the proteasome handles substrates in dynamic cellular contexts. In this work, we first review the three proteasomal DUBs and then discuss methods which can be used to monitor proteasomal degradation in mammalian cells. We then focus on the proteasomal DUB UCH37, which has been shown to specifically cleave the branched ubiquitin chain architecture. Interestingly, UCH37 associates not only with the proteasome, but also the INO80 chromatin remodeling complex. Here, we used L-azidohomoalanine (AHA) labeling and GFPu to show that UCH37 promotes the degradation of nascent proteins and a model substrate modified with branched chains. At steady state, CRISPR knockout of UCH37 resulted in several transcript-level changes in additional cellular processes, including the cell cycle. Following synchronization at the G1/S transition, UCH37 knockout was also associated with an increased number of cells in G2. Both the proteasome and the INO80 complex actively regulate the cell cycle through chromatin-associated processes. However, given its multiple modes of activity, a complete picture of UCH37 regulation in chromatin remains unclear. We therefore examined G1/S synchronized cells using both whole cell and chromatin enrichment proteomics (ChEP). Proteomics revealed a downregulation of CDK6 with UCH37 knockout, yet this effect was not associated with altered protein degradation. Interestingly, the levels of CDK6, cyclin D1 and p21 were also affected by INO80 knockdown. Furthermore, siRNA targeting INO80 resulted in a G1/S synchronization defect similar to that observed with UCH37 knockout. In addition to its role on the proteasome, INO80-associated UCH37 may therefore be involved in gene expression during the cell cycle.

DOI

https://doi.org/10.7275/30796337

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Friday, September 01, 2023

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