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Functional Analysis of Protein S-Palmitoylation Enzymes by Dahvid Davda

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Functional Analysis of Protein S-Palmitoylation Enzymes by Dahvid Davda Functional Analysis of Protein S-Palmitoylation Enzymes by Dahvid Davda A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2015 Doctoral Committee: Assistant Professor Brent R. Martin, Chair Professor Carol A. Fierke Professor Kristina I. Håkansson Associate Professor Zaneta Nikolovska-Coleska Dedication I dedicate this work to my long-time academic mentor and dear friend Dr. Tracy Simpson, who inspired me to study the life sciences. ii Acknowledgements Throughout this thesis work and my entire graduate career I have shared the comfort and had the support and encouragement of many people, to whom I am truly grateful. I will mention a few individuals here, but my apologies go out in advance to anyone who I may inadvertently missed. First and foremost, I must thank my thesis advisor, Dr. Brent Martin, for his incredible support, endless guidance and valuable instruction that were essential to my progress over the years. He has taught me many scientific lessons, the most important of which is how to be a rigorous scientist. In addition, Brent has introduced me to many exciting topics and techniques such as mass spectrometry-based proteomics and protein palmitoylation in general. His investment in me is thus greatly appreciated. I would like thank Dr. Carol Fierke for taking time to meet and mentor me in the field of protein lipidation. In addition, I wish to sincerely express my gratitude to all of my thesis committee members; in particular, I would like to acknowledge Dr. Kicki Håkansson and Dr. Zaneta Nikolovska-Colesca for upholding high-standards, recommending great experimental ideas and providing their priceless scientific point of view during all of these investigations. Special thanks go out to all Martin lab, Fierke lab and Mapp lab members, past and present, for their support, constructive discussions, helpful feedback and reagents, including those I took without their knowledge, but made sure to give back right away when they found out. While in the lab, I am thankful to have had the opportunity to mentor a handful of both graduate and undergraduate students. To the current and future members of the Martin lab, I wish you the best of luck with your investigations. Among others, extra thanks to Dr. Ruma Banerjee and her lab members for teaching me my first steady-state enzyme velocity assays among many other iii experiments. Also, thanks to Dr. Neil Marsh and his lab members for teaching me fundamental concepts in enzymology. I must acknowledge and express appreciation for the monetary contributions that I received from the University of Michigan's Rackham school of graduate studies; I have been awarded both a merit fellowship and a pre-doctoral fellowship. Without their financial support, this project would physically not have been possible. My graduate career has been a great journey and I have had the pleasure of meeting many wonderful people along the way. I want to especially thank Andrea, Mike, Aliyah and Eric from the Fierke lab, Remy from the Kopleman lab and Max from the Garcia lab for sharing many excellent experiences and sometimes providing a non- scientific social atmosphere. I would like to thank my siblings, Lucas, Elazar, Simy and Tatiana for providing me much necessary comfort, and most importantly for discussions regarding the 'big picture' of my scientific studies. Thank you for being there for me. To my parents, Itzchak and Sultana Rivka, thank you for being supportive of me during this stage of my life and all those leading up to it. Lastly, to my wife Jeannie, there are no words that I can use to describe the gratitude and appreciation I have for you. I know I would not have been able to accomplish this feat without your invaluable help, nourishment and love. I am deeply and forever indebted to you. Thank you for your companionship during this adventure; looking forward to many more. iv Preface The work herein includes both unpublished material and published research work carried out under the guidance and instruction of my thesis chair Dr. Brent R. Martin. The following describes the scientific journals to which this dissertation work was or will be submitted to in the future. Chapter 1 contains intellectual work that was contributed by me and Dr. Brent R. Martin and was published as a review for Medicinal Chemistry Communications on December of 2013. Chapter 2 consists of studies completed primarily by me and was published in ACS Chemical Biology on July of 2013. The majority of the intellectual and physical work included in Chapter 3 was contributed by me, Dr. Kristin Labby, Dr. Jeannie Hernandez and Dr. Jaimeen Majmudar. This study will be submitted in a future manuscript to Chemistry & Biology. Chapter 4 incorporates work that was accomplished by me and Dr. Jeannie Hernandez, and will be submitted to Molecular Cell as a primary research article. Appendix A contains the complete published data set for the proteomic analysis described in Chapter 2. Appendix B contains the complete unpublished data set for the proteomic analysis described in Chapter 4. The beginning of each research chapter details individuals and their contributions to the particular study, such as experiments that were conducted or intellectual input that was provided, because their efforts were critical to the success of these projects. v Table of Contents Dedication ................................................................................................................................... ii Acknowledgements .................................................................................................................... iii Preface ....................................................................................................................................... v List of Figures .......................................................................................................................... viii List of Tables ............................................................................................................................. xi List of Equations ....................................................................................................................... xii List of Appendices .................................................................................................................... xiii List of Abbreviations, Acronyms and Symbols .......................................................................... xiv Abstract.................................................................................................................................... xvi Chapter 1: Review of Protein S-Palmitoylation, Associated Enzymes, Substrates and Physiological Roles .................................................................................................................... 1 1.1 General introduction ..................................................................................................... 1 1.2 The Physicochemical Nature and Biological Implications of Protein S- Palmitoylation . 3 1.3 Tools and Techniques for Studying Protein S-Palmitoylation ....................................... 7 1.4 Functions of Protein S-Acyltransferases .....................................................................14 1.5 Pharmacological Agents for the Study of PATs ...........................................................18 1.6 Functions of S-Acyl-Protein Thioesterases .................................................................25 1.7 Pharmacological Agents for the Study of APTs ...........................................................28 1.8 Signaling Functions of Dynamic Palmitoylation ...........................................................41 1.9 Bibliography ................................................................................................................46 Chapter 2: Analysis of 2-Bromopalmitate Proteome Reactivity and Utility as a Probe for Protein S-Acyltransferases ........................................................................................................70 2.1 Abstract ......................................................................................................................70 2.2 Introduction .................................................................................................................72 2.3 Materials and Methods ................................................................................................74 2.4 Results ........................................................................................................................80 2.5 Discussion ..................................................................................................................91 2.6 Bibliography ................................................................................................................93 vi Chapter 3: Structural and Biochemical Studies of LYPLA1/2 Active Site Selectivity toward Small Molecule Inhibitors ML348 and ML349 ............................................................................96 3.1 Abstract ......................................................................................................................96 3.2 Introduction .................................................................................................................98 3.3 Materials and Methods .............................................................................................. 106 3.4 Results
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