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Structural Insight Into the Mechanisms of Activation and Substrate University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 12-12-2016 Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7 Alexandra Pozhidaeva University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Pozhidaeva, Alexandra, "Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7" (2016). Doctoral Dissertations. 1287. https://opencommons.uconn.edu/dissertations/1287 Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7 Alexandra Pozhidaeva, PhD University of Connecticut, 2016 The major part of this thesis describes studies of human ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme that regulates cellular levels of key oncoproteins and tumor suppressors. Inactivation of USP7 has recently emerged as a new approach to treatment of malignancies. However, design of potent and specific small-molecule compounds requires detailed understanding of the molecular mechanisms of USP7 substrate recognition and regulation of its catalytic activity. The goal of this work was to explore these mechanisms using solution nuclear magnetic resonance spectroscopy in combination with other methods. In our studies of USP7 substrate recognition, we structurally characterized its interaction with ICP0 protein from Herpes Simplex virus 1 and identified a novel USP7 substrate-binding site harbored within its C-terminal region. To address the question of USP7 activity regulation, we investigated its interaction with ubiquitin, which was believed to cause structural rearrangement of USP7 active site from an unproductive to a catalytically competent conformation. Surprisingly, we showed that in solution USP7 – ubiquitin interaction alone is not sufficient for activation of the enzyme as was previously postulated. Finally, we uncovered a previously unknown mechanism of USP7 inactivation by two of its known inhibitors. We found that these compounds bind to the active site of USP7 and inactivate the enzyme via covalent modification of a catalytic cysteine residue. The efficacy of the inhibitors was confirmed in cells. Altogether, these results advance our understanding of the mechanisms of substrate specificity, activation and inhibition of USP7 and open new strategies for rational structure-based drug design. Chapter 4 describes another example of using NMR spectroscopy in studies of complex biological systems. REV1 is a translesion synthesis (TLS) DNA polymerase that plays a key role in replication Alexandra Pozhidaeva – University of Connecticut, 2016 across the sites of DNA damage. Beyond its catalytic activity, its major role is to recruit other TLS polymerases to DNA lesions. The goal of this work was to structurally characterize protein – protein interactions mediated by the C-terminal region of REV1 (REV1-CT). We determined a solution NMR structure of REV1-CT alone and in complex with TLS polymerase η, and unraveled the molecular mechanism by which REV1 recognizes other Y-family TLS polymerases. Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7 Alexandra Pozhidaeva B.S., St. Petersburg State University, Russia, 2009 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2016 i Copyright by Alexandra Pozhidaeva 2016 ii APPROVAL PAGE Doctor of Philosophy Dissertation Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7 Presented by Alexandra Pozhidaeva, B.S. Major Advisor ______________________________________________________________________ Irina Bezsonova Associate Advisor ___________________________________________________________________ Dmitry Korzhnev Associate Advisor ___________________________________________________________________ Sandra Weller Associate Advisor ___________________________________________________________________ Jeffrey Hoch Associate Advisor ___________________________________________________________________ Peter Setlow University of Connecticut 2016 iii AKNOWLEDGMENTS I am extremely grateful to the many people who surrounded me during a half-decade journey towards achieving this life milestone. Here I wish to acknowledge the people without whom the completion of this dissertation would not be possible. First and foremost, I would like to express my deepest gratitude to my advisors, Dr. Irina Bezsonova and Dr. Dmitry Korzhnev, for allowing me to join their research group. It has been an honor to be their first PhD student. Despite the pressure of starting their own lab, from my first day there they showed themselves as exceptional mentors. Dmitry having a background in physics and Irina being a biologist, they taught me about science from different angles making the learning experience unique and always interesting. To Irina, your passion about protein structures was contagious and your irrepressible optimism was always a motivation for me. To Dmitry, thank you for teaching me to pay close attention to details and to be critical of the results. I would like to thank my committee members Dr. Jeffrey Hoch, Dr. Sandra Weller and Dr. Peter Setlow for their constructive dialogue, overall support and guidance. Their feedback over the years not only enabled me to take the project in new directions but also helped me develop qualities necessary for being a successful scientist. I would like to acknowledge past and present members of Korzhnev/Bezsonova lab for our fruitful scientific discussions and for the help whenever it was needed. Special thanks goes to Dr. Yulia Pustovalova for spending long nights teaching me LINUX and basics of protein structure calculation, and to Dr. Mariana Quezado for bringing a lot of laughter and cheer to the lab. I am also grateful to Dr. Mark Maciejewski for help with operating NMR spectrometers and to other iv members of the NMR super group (Dr. Adam Schuyler, Michael R. Gryk and Gerard Weatherby) for valuable comments and suggestions. If not for all the members of MBB department, my PhD experience would never be the same. Because of the diverse journal clubs, work in progress talks and seminars I now a have vast knowledge of various scientific topics that will help me to navigate through the rest of my scientific career. I would also like to thank professors Dr. Siu-Pok Yee, Dr. Henry Smilowitz and Dr. Melissa Caimano from the labs next door for insightful thoughts on the project and for non- scientific conversations reminding me about the big World outside the lab. Finally, I would like to thank my family and friends for all their love and tremendous support. To my mother Ludmila, thank you for teaching me to never give up and to always look forward. To my brother Vladislav, thank you for encouraging my interest in science since my early childhood. To many friends I acquired in Connecticut, especially to Oksana, Olena and Dina, thank you for all the wonderful time we had together. Last but not the least, to my fiancé Michael, I am deeply thankful to you for always being there for me listening, understanding and cheering. v Table of contents CHAPTER 1. Introduction ...........................................................................................................1 1.1. USP7 – a complex, pharmaceutically relevant biological system ..........................................2 1.1.1. Overview of ubiquitin – proteasome system .................................................................2 1.1.2. Deubiquitinating enzymes and their role in the ubiquitin – proteasome system ...........3 1.1.3. Introducing USP7: functions, substrates and regulation ................................................6 1.1.4. Structure of USP7 ........................................................................................................10 1.1.5. USP7 in human disease ................................................................................................13 1.1.6. Pharmaceutical potential of USP7 ...............................................................................14 1.2. NMR spectroscopy as a powerful method for studies of proteins and protein – protein interactions................................................................................................................16 1.2.1. Basics of NMR .............................................................................................................16 1.2.2. Protein structure determination using NMR spectroscopy ..........................................19 1.2.3. NMR studies of protein dynamics ...............................................................................23 1.2.4. NMR titration experiments in studies of protein –ligand interactions .........................26 1.3. Thesis objectives ...................................................................................................................29 CHAPTER 2. Structural characterization of interaction between USP7 and ICP0 E3 ligase from HSV-1 ...................................................................................................................................39 2.1. Abstract .................................................................................................................................40
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