Date of Award

Summer 7-10-2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Xuedong Liu

Second Advisor

Hang Yin

Third Advisor

Natalie G. Ahn

Abstract

The ubiquitination pathway is linked to a growing number of diseases including cancer and Parkinson's disease. Previously, the absence of robust functional assays had posed challenges in exploiting E3 ligases as therapeutic targets. The aim of this study was to use in vitro reconstituted ubiquitination systems to explore the therapeutic potential of targeting E3 ligases, in addition to gaining insight into how E3 ligases are regulated.

Telomerase activation is a rate-limiting step in carcinogenesis. However, attempts to target telomerase have been mostly unsuccessful. Thus, we targeted TRF1 - a protein that represses telomere elongation by preventing telomerase from accessing the telomeres. In Chapter 2, a reconstituted in vitro ubiquitination assay involving the E3 ligase SCFFbx4 and its substrate TRF1 was used to develop highly specific peptide inhibitors. In particular, a structure-based computational approach was used to rationally design peptides that can disrupt the E3 ligase (SCFFbx4) - substrate (TRF1) binding interface and subsequent ubiquitination. Characterization of the inhibitors demonstrates that our sequence-optimization protocol results in an increase in peptide-TRF1 affinity without compromising peptide-protein specificity.

Since it was revealed that Parkin exists in an auto-inhibited conformation, the question of how Parkin becomes activated has been under investigation. In Chapter 4, an in vitro ubiquitination assay involving the Parkin E3 ligase and the anti-apoptotic protein Bcl2 has been developed to gain insight into the activation mechanism of Parkin. We have demonstrated that phosphorylation of Parkin unlocks the auto-inhibited state of the E3 ubiquitin ligase, allowing both self-ubiquitination of Parkin and mono-ubiquitination of its substrate Bcl2.

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