Type

Text

Type

Dissertation

Advisor

Seeliger, Markus A. | Luk, Ed | Demple, Bruce | Neiman, Aaron.

Date

2017-08-01

Keywords

FKBP | Molecular biology | FRB | substrate | Ubc4 | Ubiquitin

Department

Department of Molecular and Cellular Pharmacology.

Language

en_US

Source

This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree

Identifier

http://hdl.handle.net/11401/78199

Publisher

The Graduate School, Stony Brook University: Stony Brook, NY.

Format

application/pdf

Abstract

The ubiquitin-proteasome system (UPS) regulates a myriad of signaling pathways to maintain cellular homeostasis. UPS function hinges on the conjugation of a small, highly conserved protein called ubiquitin (Ub) to target proteins. Ubiquitinated proteins can be targeted to many downstream processes such as degradation, cellular trafficking, and DNA repair. It is unsurprising, then, that aberrations in the UPS are implicated in diseases such as cancer and neurodegenerative disorders. Ub is conjugated to substrates via the sequential action of three enzymes – E1, E2, and E3. The E2 and E3 enzymes dictate the substrate specificity of the UPS. Thus, elucidating the Ub substrate spectrum of E2/E3 enzymes and their associated signaling pathways is a necessity for future pharmacological targeting. In my work, I describe a novel method to biochemically tag and identify substrates of the stress response E2 enzyme, Ubc4, in S. cerevisiae. Capitalizing on the high affinity that the protein domains FRB and FKBP have for each other only in the presence of rapamycin, I fused FLAG-tagged Ub and Ubc4 to these domains, generating the fusion proteins FRB-Ub and Ubc4-FKBP respectively. I first established that FRB-Ub and Ubc4-FKBP were active in vitro. I then developed and optimized this rapamycin-inducible system within yeast to enable spatio-temporal regulation of Ubc4-specific protein ubiquitination with FRB-Ub. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with quantitative proteomics of FLAG-affinity purified FRB-ubiquitinated substrates allowed me to validate the functionality of my system. I identified bona fide Ubc4 substrates such as Rsp5, Hht1, Itr1, and Ufd4 in rapamycin-treated samples. These substrates were identified alongside other proteins that could feasibly be tied to Ubc4 activity, though further validation is still needed. This tool sets the stage for increasing our knowledge of the substrates and signaling pathways of UPS enzymes and will ultimately inform the process of developing effective and specific therapies across different diseases. | 161 pages

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