University of Kentucky UKnowledge Theses and Dissertations--Microbiology, Microbiology, Immunology, and Molecular Immunology, and Molecular Genetics Genetics 2012 PRION CHARACTERIZATION USING CELL BASED APPROACHES Vadim Khaychuk University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Khaychuk, Vadim, "PRION CHARACTERIZATION USING CELL BASED APPROACHES" (2012). Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics. 2. https://uknowledge.uky.edu/microbio_etds/2 This Doctoral Dissertation is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Vadim Khaychuk, Student Dr. Glenn C. Telling, Major Professor Dr. Charlotte S. Kaetzel, Director of Graduate Studies PRION CHARACTERIZATION USING CELL BASED APPROACHES _______________________________________________ Dissertation _______________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Vadim Khaychuk Lexington, Kentucky Director: Glenn C. Telling, Ph.D. Professor of Microbiology, Immunology, and Molecular Genetics Lexington, Kentucky Copyright © Vadim Khaychuk 2012 ABSTRACT OF DISSERTAION PRION CHARACTERIZATION USING CELL BASED APPROACHES Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources. KEYWORDS: Prions; Cellular Permissiveness; Cellular PrPC; Protease Resistant PrPSc; Transcriptional Differences Vadim Khaychuk March 16, 2012 PRION CHARACTERIZATION USING CELL BASED APPROACHES By Vadim Khaychuk Glenn C. Telling, Ph.D. Director of Dissertation Charlotte S. Kaetzel, Ph.D. Director of Graduate Studies March 16, 2012 Date DEDICATION This dissertation is dedicated to my parents, brother and grandparents (Rita and Slava Khaychuk, Felix, Masha and David Khaychuk), who instilled in me the moral virtues of hard work and diligence, and who were unequivocally supportive throughout my scientific journey. In Loving Memory Of My Dear Cousin Anton Khlevitt 1982-201 ACKNOWLEDGEMENTS In completion of my graduate school career, I would like to express sincere gratefulness and appreciation to the many people helping me along the way. First and foremost, I would like to thank my mentor, Dr. Glenn C. Telling, for all of his guidance towards the completion of my project and keeping me focused on the primary task. Furthermore, I would like to thank him for always pushing me to achieve my best. Throughout this process, Dr. Telling became not just a mentor but also a great friend. I would also like to acknowledge and thank my committee; Dr. Alan Kaplan, Dr. Beth Garvy, Dr. Chongsuk Ryou and Dr. Hansruedi Büeler, for their dedication and commitment in helping me complete my graduate school career. I greatly appreciate the effort that was taken by Dr. Telling and my committee to help me seamlessly integrate into the graduate program upon my transfer from University of Rochester. Throughout my graduate career I have had the pleasure to work alongside wonderful lab members that I want to thank and acknowledge. To all of the Telling lab members (past and present), thank you for providing me with great friendship and assistance when I needed it. Specifically, I would like to thank Jifeng Bian, Hae-Eun Kang and Sehun Kim, Eri Saijo and Yuri Klyachkin for their friendship, technical support during critical experiments and providing me with the much-needed feedback about my research throughout the years. I would like to thank Dr. Arnold Stromberg, Dr. Peter Huggins, Dr. Anthony Athippozhy, Dr. Nigel Cooper and Xiaohong Li (Xiao) for dedicating the time, energy and effort to technically support and train me to conduct the microarray gene expression studies. iii Finally, I want to thank my family and friends for the support they have provided me throughout my graduate school career. To my parents and grandparents, I am eternally grateful for their support in my career choice and willingness to try to understand my research. I would like to thank them for everything they sacrificed to leave our home country of Ukraine to bring my brother and I to this country, which ultimately provided me with this opportunity to succeed and become the person I am today. To my brother, Felix and his family, thank you for all of your support and friendship. Lastly, I would like to thank my dearest friends Vlad and Genna Mirochnik, Ilya Vaynshteyn, Alex Nesterenko and Irene Chernyak for always being there for me, no matter what. iv TABLE OF CONTENTS Acknowledgements………………………………………………………………………iii List of Tables…………………………………………………………………………......vi List of Figures ...................................................................................................................vii Abbreviations....................................................................................................................viii Chapter 1: General Introduction to Prion Biology Introduction………………………………………………………………..............1 Section I: General Introduction to Prion Biology………………………....1 Section II: Experimental Approaches To Analyze Prions……….............27 Section III: PrP Interacting Molecules.......................................................38 Dissertation research………………………………………………………..........70 Chapter 2: Materials and methods.....................................................................................72 Chapter 3: Characterization of Prion Strains in Cell Culture Systems Introduction………………………………………………………………............93 Results……………………………………………………………………............99 Section I: Prion Species Barriers,