Mechanistic Studies of the E3 Ubiquitin Ligases SCF(Fbx4) and Parkin

Date____________________ The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. Thesis directed by Professor Xuedong Liu 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 SCF Fbx4 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 (SCF Fbx4)-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 iv 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. v Acknowledgements I would like to thank my advisor, Dr. Xuedong Liu, for the years of support and guidance. His optimism and passion for science has encouraged me throughout the years. I am deeply grateful. I thank my committee members Drs. Wang for your friendship. Graduate school wouldn't have been so much fun without you. I would like to thank our undergraduate research assistant Timothy Kopper for his assistance. I thank my parents, Hwajin Lee and Dongho Lee. I thank my sister Ada Younglim Lee. Last but not least, I thank my husband John Y. Kim for always being on my side.