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Signature Redacted Certified By: Ronald T Endogenous and Chemical Modifications of Model Proteins by Valerie T. Ressler B.A. Chemistry Macalester College, 2011 M.S. Chemistry College of William & Mary, 2013 Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY at the Massachusetts Institute of Technology February 2019 @ 2019 Massachusetts Institute of Technology. All rights reserved. Signature Signature of Author: redacted____ Department of Chemistry January 14, 2019 -Signature redacted Certified by: Ronald T. Raines Firmeni Pro essor of Chemistry hesis Supervisor Signature redacted Accepted by: Robert W. Field OFTECHNOWGO Haslam and Dewey Professor of Chemistry Chairman, Departmental Committee on Graduate Students MAR 21 2019 LIBRARIES ARCHIVES 1 This doctoral thesis has been examined by a committee of professors from the Department of Chemistry as follows: Signature redacted Matthew D. Shoulders Whitehead CD Associate Professor Thesis Committee Chair Signature redacted Ronald T. Raines Firmenich Professor of Chemistry I A Thesis Supervisor Signature redacted Laura L. Kiessling Ne~ovartis Professor of Chemistry Thesis Committee Member 2 Endogenous and Chemical Modifications of Model Proteins by Valerie T. Ressler Submitted to the Department of Chemistry on January 15, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry Abstract Protein modifications are ubiquitous in nature, introducing biological complexity and functional diversity. Of the known post-translational modifications, glycosylation is one of the most common and most complex, yet some of the biological implications of this modification remain poorly understood. The development of chemical tools to mimic these modifications is helping to elucidate their biological roles and improve the range of biopharmaceuticals. To probe the biochemistry of endogenous glycosylation and to test the efficacy of novel synthetic modifications, tractable protein scaffolds are needed. Previously, members of the pancreatic-type ribonuclease (ptRNases) superfamily have been utilized as model protein scaffolds. They are a class of highly conserved, secretory endoribonucleases that mediate diverse biological functions through the cleavage of RNA. The prototypical family homolog, human ribonuclease 1 (RNase 1), has been observed as a differentially glycosylated protein in vivo and been shown to tolerate a wide range of chemical manipulations. It has also emerged as an ideal candidate for protein-based drug therapy. The goal of this thesis is to showcase the biological potential of RNase 1 as a model endogenously glycosylated protein and as a protein payload for evaluating intracellular delivery systems. 3 In CHAPTER 1, I summarize the current knowledge about ptRNases including their biochemical characterization, conservation of N-glycosylation, and therapeutic potential. RNase 1 possesses three N-glycosylation sites giving rise to enormous heterogeneity in biological samples, with unknown implications. In CHAPTER 2, I demonstrate that glycosylation of RNase 1 enhances protein stability and attenuates enzymatic activity. In CHAPTER 3, I utilize a previously developed diazo compound to enhance delivery of a therapeutically relevant RNase 1 variant. The modification is shown to be reversed upon entry into the cell, presenting a novel approach for delivering native, functional proteins to the cytosol. Intracellular delivery of another model protein, Cytochrome C (CytoC), has shown therapeutic promise as well. In CHAPTER 4, I demonstrate that synthetic glycosylation with a large, monofunctionalized dextran conveys CytoC into the intracellular space, triggering apoptosis. Finally, CHAPTER 5 outlines future directions for the study of RNase 1 glycosylation and expanding the utility of the established diazo and dextran-based delivery systems. Taken together, this thesis explores a wide variety of protein modifications, demonstrating biochemical effects of endogenous glycosylation and enhanced delivery of protein payloads with chemical tools. Thesis Supervisor: Ronald T. Raines Title: Firmenich Professor of Chemistry 4 Acknowledgments I would like to express my sincere thanks to the many people who have encouraged me and supported me on my journey through graduate school. First and foremost, I would like to thank Professor Ronald Raines for allowing me to be a part of his amazing research group and giving me the opportunity to pursue research in his laboratory. My time in the Raines lab has allowed me to work on a wide range of projects with many brilliant people. As a result, I have evolved into a confident, independent scientist and thinker. I have been fortunate to be a part of several successful projects that would not have been possible without my fantastic collaborators. Thanks to Henry Kilgore, Kalie Mix, and Wen Chyan for their insightful contributions and hard work that helped drive these projects forward. I also want to thank my predecessor Rob Presler for establishing the foundation for the glycosylation project. I have to thank my undergraduate assistant, Jamie Wood, for her dedicated work with enzyme activity assays and her organization skills that helped keep our biochemistry experiments moving seamlessly. I have to thank the Raines lab as a whole, both past and present, who have routinely given me their time and attention. I especially have to thank Ian Windsor, Trish Hoang, Jim Vasta, Robert Newberry, and Caglar Tanrikulu for their thoughtful discussions and eagerness to lend a helping hand. I am grateful to have been a part of such a wonderful lab that has encouraged me to grow personally and professionally. It has been a pleasure to be a part of such a fun, close-knit group. I would like to thank the Department of Chemistry at the University of Wisconsin for their assistance and guidance in starting my graduate career. Brian Esselman and Nick Hill were instrumental in cultivating my passion for mentoring and teaching. I would also like to thank the 5 Department of Biochemistry at the University of Wisconsin for their support of my work and funding me through a William H. Peterson Fellowship. The move to Massachusetts Institute of Technology was monumental and helped along by many people. The Kiessling lab members have been a great source of advice, encouragement, and laughter as we reassembled our labs and found our way in this new space. Brian Pretti and Jennifer Weisman welcomed us to MIT and have since been wonderful resources as we re- established the lab and joined the Chemistry Department. I am particularly grateful to Professor Laura Kiessling and Professor Matt Shoulders for their flexibility in acting as my new thesis committee at MIT. I appreciate their time and helpful suggestions throughout my time here. I have to thank Alex Justen, Greg Jones, Will Czaplyski, Kaila Margrey, and Emily Garnett for being incredible friends and sources of comfort during the last years of graduate school - you have provided endless amounts of encouragement and for that I am forever grateful. My sincere thanks to my family, Tripps, Resslers, and Frybergers, your support has always allowed me to follow my dreams, wherever they may lead. I would especially like to thank my mom and brother, Miles, for their herculean efforts at Southwest Airlines and American Airlines that allowed my husband to log more than 25,000 air miles on trips to visit me in Boston (the circumference of the Earth is 24,901 air miles). Finally, thank you to Daniel Ressler for his unwavering support and patience. 6 Table of Contents: A bstract.....................................................................................................................................3 Acknowledgm ents...............................................................................................................................5 List of figures and schem es................................................................................................................10 List of tables......................................................................................................................................12 Abbreviations....................................................................................................................................13 Chapter 1 ................................................................................................................................. 15 Understanding Endogenous and Chemical Protein Modifications .................................. 15 1.1 Introduction.................................................................................................................................16 1.2 The pancreatic-type ribonuclease superfam ily ........................................................................... 17 1.2. 1: Biochemical characteristicsof ptRNases ........................................................................................ 17 1.2.2: Known biologicalfunctionsof the human ptRNase superfamily........................................................ 18 1.3 Hum an ribonuclease 1 ................................................................................................................. 20 1.3.1: Secretion andglycosylation of RNase 1 in vivo ............................................................................... 20 1.3.2: RNase 1 homologs across vertebrate species ................................................................................. 21 1.3.3:
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