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Redefining the Scope of Prenylation Syracuse University SURFACE Dissertations - ALL SURFACE August 2018 REDEFINING THE SCOPE OF PRENYLATION: DISCOVERY OF “FORBIDDEN” SUBSTRATE RECOGNITION AND DEVELOPMENT OF METHODS UTILIZING PRENYLATED PROTEINS Melanie Jane Blanden Syracuse University Follow this and additional works at: https://surface.syr.edu/etd Part of the Physical Sciences and Mathematics Commons Recommended Citation Blanden, Melanie Jane, "REDEFINING THE SCOPE OF PRENYLATION: DISCOVERY OF “FORBIDDEN” SUBSTRATE RECOGNITION AND DEVELOPMENT OF METHODS UTILIZING PRENYLATED PROTEINS" (2018). Dissertations - ALL. 911. https://surface.syr.edu/etd/911 This Dissertation is brought to you for free and open access by the SURFACE at SURFACE. It has been accepted for inclusion in Dissertations - ALL by an authorized administrator of SURFACE. For more information, please contact [email protected]. ABSTRACT Post-translational modifications play a central role in controlling biological function and cell behavior through changes in protein structure, activity, and localization. Prenylation is one such modification wherein a 15- or 20-carbon isoprenoid group is attached to a cysteine residue near the C-terminus of a substrate protein by one of three enzymes: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I) or protein geranylgeranyltransferase type II (GGTase-II, also known as Rab GGTase). These covalent modifications can aid in protein association with cellular membranes, with this localization necessary for function of many prenylated proteins. FTase and GGTase-I have been proposed to recognize a four amino acid “Ca1a2X” C-terminal sequence based on biochemical, structural, and computational studies of these enzymes. However, recent genetic screening studies in yeast suggest the potential for FTase to prenylate sequences of the form -C(x)3X, with four amino acids downstream of the cysteine residue to be prenylated. The work herein begins to define the sequence scope for this -C(x)3X motif, establishes the biological relevance of this new class of prenyltransferase substrates in cells, and supports future investigation of the impact of these non- canonical prenylated proteins on cell behavior and biological function. With the discovery of new -C(x)3X recognition motifs in prenylation, new methods with which to identify proteins capable of being prenylated are required. To this end, we have explored the use of engineered FTase variants, specifically RL FTase, selected for the ability to prenylate substrate sequences that are unreactive with WT FTase. Combining this engineered FTase variant with functionalized FPP analogues yields a bioorthogonal selective technique for isolating target proteins, even in the presence of other prenyltransferase substrate proteins in cell lysates. The value of this method is demonstrated by selective pulldown of model fluorescent proteins in bacterial lysates in the presence of competitor proteins. The selectivity of FTase- catalyzed prenylation and the minimal size of the C-terminal FTase recognition motif render this approach applicable to a wide range of target proteins. A second quantitative method introduced here is Protein-Lipidation Quantitation (PLQ); a new method that can simultaneously measure the amounts of a non-lipidated substrate protein and its lipidated product in a cellular context. In PLQ, use of a fluorescent protein fused to the substrate under investigation allows for quantitative detection of both the non-lipidated substrate and the lipidated product. Upon prenylation in cells, the substrate and the product in these cell lysates are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. This work demonstrates the usefulness of PLQ both in principle and in application with its ability to confirm a link between a mutation in the p53 tumor suppressor gene and cellular prenylation activity. The quantitative capabilities of PLQ will allow researchers to address previously unanswered hypotheses regarding protein lipidation and its roles in cellular regulation and biological function. REDEFINING THE SCOPE OF PRENYLATION: DISCOVERY OF “FORBIDDEN” SUBSTRATE RECOGNITION AND DEVELOPMENT OF METHODS UTILIZING PRENYLATED PROTEINS by Melanie Jane Blanden M. Phil., Chemistry, Syracuse University, 2013 B.S., Forensic Science, Bay Path College, 2011 Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Syracuse University August 2018 Copyright © by Melanie Jane Blanden 2018 All Rights Reserved ACKNOWLEDGEMENTS In recognizing the people who have helped me develop into the scientist I am today, I must first thank Dr. James Hougland for his support and guidance during my time at SU. His ability to challenge me and his belief that my abilities are greater than I realize, have pushed me beyond what I thought capable and made me a better scientist and person. Without his level of understanding and support for my desire to become both mother and scientist during my years in graduate school, I could have not accomplished the difficult task of the work-life balance which comes along with it. I am also extremely grateful for his help, trust, and support in realizing my dream of becoming a college professor. This last year was a balancing act and it was our professional dynamic and understanding that allowed me to "have it all" and I will always be grateful. I would also like to thank Dr. Michael Sponsler and Dr. John Chisholm for reviewing my work throughout the years and providing their support. I would also like to thank Dr. Shikha Nangia, Dr. John Franck, and Dr. Carlos Castañeda for serving on my thesis committee and providing valuable feedback and discussion. I am extremely grateful to my lab family: Susan, Joe, Michelle, Soumya, Rosie, Liz, Kayleigh, Maria, Tasha, Sudhat, Jacob, and Mariah. Susan, Joe, and Michelle taught me the basic lab skills I needed to succeed but everyone of you has made coming into the lab everyday a fun, rewarding experience. Thank you for the countless smiles, laughs, and memories. I would also like to say thanks to my colleagues at Utica College for their support during a busy year, for liking me enough to keep me, and for gifting me the many cups of coffee I desperately needed. As an undergraduate student I found the unending support of professors such as Dr. Gina Semprebon, and Dr. Edward Bernstine, but especially of Dr. Hsiang-ching Kung. Dr. Kung saw something special in my abilities and passion for chemistry and his belief in me inspired a belief v in myself. He has been an integral part of my journey and if he has ever wondered if he has changed a student's life, he has. I do not have enough words to acknowledge and thank my husband, Adam, for everything he has done for me since we set out on our life together. We have made graduate school, parenthood, marriage, and careers work because of our ability to be a team and the sacrifices I know you made. God blessed me when I met you in Boston and for the innumerable ways you have helped me grow into a strong, confident person. Thank you for being my other half and for bearing the weight of so many responsibilities these past years. Thank you for pushing me because you knew what I was capable of. I also need to thank my daughter, Leah, not for making graduate school easier (trust me...you made it so much harder) but for igniting a motivation in me. I worked harder knowing you were watching me and that makes this that much more rewarding. I love you Miss B, and I can’t wait to see what you accomplish in this world. Thank you to my parents, Mary and Stephen, for building me into who I am today and for being incredible role models. I have been able to overcome the challenges I have faced because I learned courage and perseverance from you. I know you are my biggest fans and I love you very much. Thank you also to my siblings: Brian, Lindsay, and Luke for all our incredible memories and antics. It is a blessing to have siblings you can call your friends. Thank you to my grandparents Fran Card, and Mel and Dorothy Decker for always supporting me. Thank you especially to my Grandma Frannie for the hour-long phone calls about everything from day-to- day nonsense to the frustrations of troubleshooting my research. Last but not least, thank you to the family I married into. Marie and Wally Blanden are the most incredibly supportive in-laws I could have imagined, and I appreciate them so much. Thanks for the homecooked meals, being heroes in a crisis...many of them, and the never-ending love you give Adam, Leah, and myself. vi TABLE OF CONTENTS Chapter 1: Introduction 1.1 Post-translational modifications...............................................................................1 1.2 The prenylation pathway..........................................................................................6 1.3 Interactions and specificity between prenyltransferases and target proteins.........10 1.4 Biological significance of prenylation...................................................................21 1.5 Importance and objectives.....................................................................................24 1.6 References for chapter 1........................................................................................27 Chapter 2: Investigation of -C(x)3X sequence recognition by FTase and GGTase-I in vitro and in cell 2.1 Introduction for chapter 2......................................................................................53
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