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Structure, Function, and Inhibition of Protein Depalmitoylases by Sang

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Structure, Function, and Inhibition of Protein Depalmitoylases by Sang Structure, Function, and Inhibition of Protein Depalmitoylases by Sang Joon Won A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2018 Doctoral Committee: Assistant Professor Brent R. Martin, Chair Professor Philip C. Andrews Associate Professor Jolanta Grembecka Professor John J. G. Tesmer Sang Joon Won [email protected] ORCID iD: 0000-0002-8148-7652 Dedication I dedicate this dissertation to all my teachers and mentors. ii Acknowledgements First, I would like to thank my family for their continuous support throughout my journey so far. They are the best mentors who will always listen to me and give honest advice. I would not have arrived in America without their support and trust in my abilities. Thanks to them, I always felt confident in challenging the new chapters in my life so far. I would like to thank my mentors and teachers at the University of Wisconsin- Madison, who first introduced me to the real science. Professor Michael Cox and Dr. Marielle Gruenig are the people I always admired as scientists and as human being. They became my role models and inspired me to continue my study in graduate school I also thank Dr. Venki Ramakrishnan and Dr. Alexey Amunts for supervising me at the MRC-Laboratory of Molecular Biology, Cambridge. They are the first ones to introduce me to the structural biology. From my two months of experience, I have learned the true dedication of the top-level scientists. I would like to thank my committee members Prof. Philip Andrews, Prof. Jolanta Grembecka, and Prof. John Tesmer for their critical feedback and putting me on the right path to graduation. I rotated in Prof. Tesmer’s lab and with Dr. Alisa Glukhova’s help, I really enjoyed my first lab experience here in Michigan. I have learned a lot from Prof. Tesmer with his ability to provide critical insight into problems. I would like to thank Prof. Brent Martin for being my mentor and advisor. He has impressed me with his knowledge across diverse disciplines in science. His ability to think critically with open-minded attitude has always had positive influence on me. I feel I got trained as a scientist who can be critical of my own data and reach out beyond the comfort-zone of my current abilities. Thanks to him, I got my first experience in many disciplines in science as this dissertation will show. I am very fortunate to be in his lab. I got to experience the true chemical biology, which was my primary reason for coming to this university. iii I would like to thank the Rackham Graduate school providing me with the pre- doctoral fellowship and travel grants. Finally, I would like to acknowledge the past and present lab members of the Martin lab for providing wonderful support. To mention a few, I would like to thank Dr. David Davda for teaching me technical skills in biochemistry and Dr. Chris Tom for the organic synthesis. Dr. Jaimeen Mujmudar for very helpful advice on chemical synthesis as well as proteomic analysis. Melanie Cheng See Kit and Sarah Haynes for their support. These people made my journey in Michigan easier and it will never be forgotten. iv Table of Contents Dedication.......................................................................................................................... ii Acknowledgements ......................................................................................................... iii List of Figures ................................................................................................................. vii List of Tables ..................................................................................................................... x List of Appendices .......................................................................................................... xi Abstract ........................................................................................................................... xii Chapter 1 : Protein Depalmitoylases ............................................................................. 1 Abstract ............................................................................................................................. 1 Introduction ...................................................................................................................... 2 Assays for depalmitoylation ............................................................................................... 4 Palmitoyl protein thioesterase 1 ........................................................................................ 8 Acyl protein thioesterases .................................................................................................. 9 Structural insights to APT enzymes ................................................................................... 16 ABHD17 family thioesterases ........................................................................................... 20 Conclusions...................................................................................................................... 23 Individual contributions ................................................................................................... 25 Chapter 2 : Molecular Mechanism for Isoform-selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2) ..................................................................... 26 Abstract ........................................................................................................................... 26 Introduction .................................................................................................................... 27 Results ............................................................................................................................. 28 Discussion ........................................................................................................................ 43 Materials and Methods .................................................................................................... 46 Acknowledgements ......................................................................................................... 50 v Individual contributions ................................................................................................... 50 Chapter 3 : Affinity-based Selectivity Profiling of an In-class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2................................................ 52 Abstract ........................................................................................................................... 52 Introduction .................................................................................................................... 53 Results ............................................................................................................................. 55 Discussion ........................................................................................................................ 63 Materials and Methods .................................................................................................... 64 Individual contributions ................................................................................................... 67 Chapter 4 : Temporal Profiling Establishes a Dynamic S-palmitoylation Cycle .... 68 Abstract ........................................................................................................................... 68 Introduction .................................................................................................................... 69 Results ............................................................................................................................. 70 Discussion ........................................................................................................................ 83 Materials and Methods .................................................................................................... 84 Individual Contributions ................................................................................................... 90 Acknowledgments ........................................................................................................... 90 Chapter 5 : Conclusions and Future Directions ......................................................... 95 Conclusions...................................................................................................................... 95 Future directions to analyze APT function ........................................................................ 96 Future directions towards understanding dynamic S-palmitoylation ............................... 100 Appendices ................................................................................................................... 105 References .................................................................................................................... 118 vi List of Figures FIGURE Figure 1.1 Graphical abstract showing depalmitoylation event mediated by a thioesterase. .............................................................................................................. 2 Figure 1.2 Cellular pathways of protein depalmitoylation. ......................................... 4 Figure 1.3 APT inhibitor selectivity index. .................................................................. 13 Figure 1.4 APT2 inhibition restores Scribble plasma
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