Award Date

12-15-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Committee Member

Gary Kleiger

Second Committee Member

Ernesto Abel-Santos

Third Committee Member

Hui Zhang

Fourth Committee Member

Laurel Raftery

Number of Pages

159

Abstract

Ubiquitylation is a cellular regulatory system found in all eukaryotic cells, which has managed to find a role in most pathways imaginable. The system works fundamentally through the ligation of a small protein known as ubiquitin onto a substrate. Depending on the context of the ubiquitin ligation, the substrate can be directed towards a number of cellular fates, the best-studied being degradation of the substrate. While originally thought of as a signal for cellular disposal units to degrade aberrant proteins, we now know that ubiquitin plays a highly nuanced role in cellular epistasis, controlling everything from the cell cycle to the immune response.

Of the many enzymes involved in transferring ubiquitin molecules to and from targeted substrates, the cullin-RING ubiquitin ligases (CRLs) stand out for their particular breadth. Hundreds of CRLs exist in human cells owing to their modular structure, which in turn allows them to regulate an even greater number of substrates. They have also been targets of a number of different drug therapies, due to their involvement in the cell cycle and cancer. However, there are many vital gaps in how they function.

Particularly, CRLs function with a number ubiquitylating enzymes, referred to here as CRL partners. The first discovered of these, Cdc34, has been prominently studied for decades, but particular aspects of its molecular mechanism for transferring ubiquitin to substrates were not yet known. Further complexity was added when other CRL partners were discovered to also function in addition to Cdc34. Promising models suggested that these CRL partners could complement the activity of Cdc34 to maximize CRL turnover of substrate, but relatively little work had been done to study this system under the consideration of physiological conditions and concentrations.

Therefore, the central aims of the studies within the planned dissertation are three-fold. First, by designing and refining current assays to set a guideline by which to measure complex, multi-component reactions. Second, by uncovering the molecular mechanism of Cdc34’s catalytic activity, so that it can be understood in the context of full CRL activity. Lastly, by determining how CRLs and their partners operate in the context of a living cell. For these efforts, we discovered a new molecular mechanism of Cdc34 activity, a new understanding of how CRL partners are balanced and used in the context of physiological CRL ubiquitylation pathways, and the unexpected complementary role of a little-studied CRL partner.

Keywords

cdc34; cullin-ring; enzyme; kinetics; ubiquitin

Disciplines

Biochemistry

File Format

pdf

File Size

14.6 MB

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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