Award Date

8-1-2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry and Biochemistry

First Committee Member

Gary Kleiger

Second Committee Member

Ernesto Abel-Santos

Third Committee Member

Hong Sun

Fourth Committee Member

Nora Caberoy

Number of Pages

47

Abstract

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system (UPS). In the UPS, ubiquitin-conjugating enzymes and ubiquitin ligases append poly-ubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount since a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, the reaction catalyzed by nuclear ubiquitin ligase San1 was reconstituted in vitro. Our results demonstrate that San1 can function with 2 ubiquitin-conjugating enzymes, Cdc34 and Ubc1. While Cdc34 and Ubc1 are both sufficient for promoting San1 activity, San1 functions preferentially with Ubc1, including when both Ubc1 and Cdc34 are present. Notably, a homogeneous peptide that mimics a misfolded PQC substrate was developed and enabled quantification of the kinetics of San1-catalyzed ubiquitylation reactions. We discuss how these results may have broad implications for the regulation of PQC-mediated protein degradation.

Keywords

Protein Degradation; Protein Misfolding; Protein Quality Control; Ubiquitin; Ubiquitin-Conjugating Enzyme; Ubiquitin Ligase

Disciplines

Biochemistry | Chemistry

File Format

pdf

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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