Peptide-Based Probes to Monitor Cysteine-Mediated Protein Activities

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Peptide-Based Probes to Monitor Cysteine-Mediated Protein Activities Peptide-Based Probes To Monitor Cysteine-Mediated Protein Activities Author: Nicholas Pace Persistent link: http://hdl.handle.net/2345/bc-ir:104128 This work is posted on eScholarship@BC, Boston College University Libraries. Boston College Electronic Thesis or Dissertation, 2015 Copyright is held by the author, with all rights reserved, unless otherwise noted. Boston College The Graduate School of Arts and Sciences Department of Chemistry PEPTIDE-BASED PROBES TO MONITOR CYSTEINE-MEDIATED PROTEIN ACTIVITIES Dissertation by NICHOLAS J. PACE submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2015 © copyright by NICHOLAS J. PACE 2015 Peptide-Based Probes to Monitor Cysteine-Mediated Protein Activities by Nicholas J. Pace Thesis Advisor: Eranthie Weerapana Abstract Cysteine residues are known to perform an array of functional roles in proteins, including nucleophilic and redox catalysis, regulation, metal binding, and structural stabilization, on proteins across diverse functional classes. These functional cysteine residues often display hyperreactivity, and electrophilic chemical probes can be utilized to modify reactive cysteines and modulate their protein functions. A particular focus was placed on three peptide-based cysteine-reactive chemical probes (NJP2, NJP14. and NJP15) and their particular biological applications. NJP2 was discovered to be an apoptotic cell-selective inhibitor of glutathione S-transferase omega 1 and shows additional utility as an imaging agent of apoptosis. NJP14 aided in the development of a chemical-proteomic platform to detect Zn2+-cysteine complexes. This platform identified both known and unknown Zn2+-cysteine complexes across diverse protein classes and should serve as a valuable complement to existing methods to characterize functional Zn2+-cysteine complexes. Finally, NJP15 was part of a panel of site-selective cysteine- reactive inhibitors of protein disulfide isomerase A1 (PDIA1). These inhibitors show promise in clarifying the unique and redundant properties of PDIA1’s dual active-sites, as well as interrogating the protein’s role in cancer. Together, these case studies illustrate the potential of cysteine-reactive chemical probes to modulate protein activities, interrogate biological systems, and aid in the development of powerful therapeutic drugs. Acknowledgements I would like to begin by thanking my advisor, Professor Eranthie Weerapana. I cannot thank you enough for guidance over these years. From a scientific stand-point, you taught me how to solve problems and think critically and independently. Your patience and leadership was exemplary, and led to both a productive and pleasurable work environment for all of us. I truly enjoyed the opportunity to work in your lab. I would also like to extend a sincere thanks to all of my thesis committee, Mary Roberts, Jianmin Gao, and Abhishek Chatterjee for all your help and advice of the years. Thank you to all of my collaborators. A special thanks goes to Sharon Louie, Dr. Mela Mulvihill, and Dr. Daniel Nomura from University of California Berkeley. Thank you for hosting me, teaching me countless techniques, and even letting me join in on a group camping trip! Additional thanks to Kimberly Miller from the Biology Department at Boston College for helping me with the RT-PCR experiments over the past six months. Thank you to all the members of the Weerapana lab past and present: Tyler, Kyle, Lisa, Shalise, Dan, Ranjan, Masahiro, Haley, Julie, Yani, and Alex. You have all been such a pleasure to work with and learn from, and I truly enjoyed coming to lab each day. More than lab-mates you are all great friends. I will miss our “Saturday Only Playlists,” Roggie’s and Joey’s nights, and constant sports conversations (and arguments). When I joined the newly found Weerapana lab almost five years ago, a small group of us assisted Eranthie in setting up the new lab, optimizing protocols, and learning countless techniques. A special thanks needs to be extended to Julie, Yani, and Alex. You three have been instrumental to my success in graduate school and there is no way I could have i succeeded without all your help. I could not think of three better people to have worked with these past years. Thank you to all the lab technicians I have encountered over the years: John, TJ, Marek, and Bret. Thank you to all the support staff at Boston College. Steve, Ginny, Dale, Lynne P., Lynne O., Ian, Jen, Lori, Howard, Richard, and Bill “Success” Fogerty. I truly appreciate your tireless efforts and patience that allowed me to maintain focus on my studies. Your constant optimism and support was truly appreciated. Thank you to all the friends at BC I have made over the years. There are way too many of you to name, but you all have been great and I truly appreciate our friendship. Special thanks to the “Chemistry Bro’s,” keep drinking chocolate milk. Finally, I’d like to thank my family and friends outside of Boston College. My friends from SJP and Stonehill College are truly amazing. I am extremely close with my family (and future in-laws), and I cannot thank them enough for their support over these years. I special thanks has to be extended to my brother and sister for their support over these years. It is nice having siblings to discuss science with, and makes for interesting holiday conversation. I also need to thank my parents. They are two of the most self-less people I have ever encountered. Every decision they have ever made is always with me, my brother, or my sister in mind, and I truly cannot describe how much I have appreciated your love and support these years. Lastly, I would like to thank my fiancée and future wife, Amanda. You have taught me so much over these years, have been so patient with me, and always keep me grounded. This is just as much your accomplishment as it is mine. I cannot thank you enough. ii Table of Contents Acknowledgements .............................................................................................................. i Table of Contents ............................................................................................................... iii List of Figures .................................................................................................................... vi List of Tables ..................................................................................................................... xi List of Abbreviations ....................................................................................................... xiii Chapter 1. Introduction...................................................................................................... 1 Cellular roles of reactive cysteines .................................................................................. 3 Redox-catalytic cysteine residues .................................................................................... 5 Cysteine residues as catalytic nucleophiles ..................................................................... 7 Metal-binding cysteine residues .................................................................................... 10 Cysteine residues as regulators of protein functions ..................................................... 12 Conclusions ................................................................................................................... 16 References ..................................................................................................................... 17 Chapter 2. A peptide-based inhibitor of GSTO1 that selectively targets apoptotic cells 25 Introduction ................................................................................................................... 26 Results and Discussion .................................................................................................. 35 Synthesis of cysteine-reactive peptide library ............................................................ 35 Evaluation of the peptide inhibitor library ................................................................. 37 Identification of the protein target of NJP2 ................................................................ 39 Application of NJP2 as an apoptotic cell-selective inhibitor and imaging agent....... 44 Conclusions ................................................................................................................... 49 iii Acknowledgements ....................................................................................................... 50 Experimental procedures ............................................................................................... 51 References .................................................................................................................... 68 Chapter 3. A chemical-proteomic platform to identify zinc-binding cysteine residues .. 77 Introduction ................................................................................................................... 78 Overview .................................................................................................................... 78 Structural Zn2+-cysteine complexes ........................................................................... 79 Catalytic Zn2+-cysteine complexes ............................................................................. 81 Regulatory Zn2+-cysteine complexes ......................................................................... 83 Inhibitory Zn2+-cysteine
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