Date of Award

2019

Embargo Period

7-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Shaun Olsen

Second Advisor

J. Alan Diehl

Third Advisor

Christopher Davies

Fourth Advisor

Steven Carroll

Fifth Advisor

Steven Rosenzweig

Abstract

Cdc34 is an essential E2 ubiquitin-conjugating enzyme that regulates the cell cycle at the G1-S checkpoint. It has been implicated in several disease processes, primarily cancer, and has become a target for therapeutic development. As an E2, Cdc34 interacts with an E1 ubiquitin-activating enzyme and a family of SCF E3 ligases to attach ubiquitin molecules to target proteins. Moreover, Cdc34 is known to specifically select Lys48 on the end of a growing polyUb chain to Ub attachment, resulting in Lys48-linked Ub chain extension and subsequent proteasomal degradation of the target. Despite decades of investigation, the structural basis for Cdc34 activities in the cell is still not resolved. A better understanding of Cdc34 interactions with ubiquitin and its E1 and E3 enzymes partners may provide the groundwork for more successful therapeutic development in the future. Thus, in these studies, we have combined x-ray crystallography, biochemistry, yeast genetics, NMR, and mammalian cell studies to answer remaining questions regarding Cdc34. We have elucidated the molecular basis for Cdc34 recognition by its E1 enzyme, including conformational changes by the E1 and Cdc34. Further, we have determined two structures of Cdc34 in complex with Ub in different conformations that may represent two distinct mechanisms for Cdc34 activity in the cell. Finally, we have laid the groundwork for important structural studies of Cdc34 interactions with its family of E3 ligases that will build on the insights from our Cdc34~Ub thioester structures. Altogether, our investigations have provided a large step forward in understanding the molecular underpinnings of Cdc34 activities in the cell.

Rights

All rights reserved. Copyright is held by the author.

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