Biochemical Interrogation of Rare Cystic Fibrosis Mutations Informs Strategies for Future Therapeutic Intervention by Steven Victor Molinski A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Biochemistry University of Toronto © Copyright by Steven Victor Molinski (2016) 1 Biochemical Interrogation of Rare Cystic Fibrosis Mutations Informs Strategies for Future Therapeutic Intervention Steven Victor Molinski Doctor of Philosophy Department of Biochemistry University of Toronto 2016 Abstract There are over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR ) gene associated with Cystic Fibrosis (CF) disease, and to date, the two recently FDA- approved drugs Kalydeco TM and Orkambi TM have been effective in rescuing functional expression of the two most common CFTR mutations, G551D and ΔF508, respectively, representing approximately 90% of the patient population worldwide. However, potential efficacy of these therapies for the remaining ~2000 mutations is unknown. Thus, biochemical characterization of each rare variant is needed to assess potential clinical benefit. My thesis attempts to understand molecular defects associated with less common CFTR mutations, mainly c.3700A>G (causing abberant splicing and subsequent translation of ΔI1234_R1239- CFTR) and c.2052_2053insA (Q685TfsX4-CFTR), with the goal of determining whether current CF therapies (specifically designed for G551D and ΔF508) could be used for these rare variants. Additionally, my work aims to identify unique molecular aberrations for these rare, mutant CFTR proteins in order to facilitate development of mutation-specific therapeutics. Through a collaborative initiative involving clinicians, biochemists and biophysicists, we found that ΔI1234_R1239-CFTR shared certain biochemical attributes with ΔF508-CFTR, however contained intrinsic defects which differed significantly (i.e. poorer interdomain assembly ii between the amino- and carboxy-termini, and decreased functional responses to cAMP agonists, CFTR correctors and potentiators). When CFTR activity was evaluated in nasal epithelial cultures from siblings homozygous for c.3700A>G, we likewise found that ΔI1234_R1239-CFTR responded poorly to small molecules. For Q685TfsX4-CFTR, we found that this variant was prematurely truncated, and therefore did not respond to CFTR-specific therapies. However, a proof-of-concept in vitro strategy to ‘skip’ the exon containing the c.2052_2053insA mutation led to an incomplete, yet partially mature CFTR molecule which exhibited channel activity, albeit this function was unregulated. In parallel to these studies, we generated and functionally characterized several other rare CFTR mutations using novel mutagenesis and high-throughput functional screening methods, allowing for rapid identification and stratification of genotypes that could potentially benefit from current CF therapies. Furthermore, our efforts described here aim to facilitate future mutation-specific CF drug discovery, as well as a ‘personalized medicine’ approach to assist in the development of novel strategies to combat this fatal genetic disease. iii Statement of Co-authorship This thesis is based on research conducted by Steven V. Molinski under the supervision of Dr. Christine E. Bear. All data were generated, analyzed and interpreted by Steven V. Molinski with the assistance of Dr. Christine E. Bear as well as many other colleagues; specific contributions to this thesis by each individual are acknowledged at the beginning of each chapter and in the captions of relevant figures. iv Acknowledgements First and foremost, I would sincerely like to thank my supervisor, Dr. Christine Bear, for providing me with the opportunity to work in her lab, as well as for allowing me to have the intellectual freedom to pursue novel ideas. I would also like to thank Christine for providing the guidance and training to become a well-rounded scientist. Christine has passionately led the pursuit of acquiring therapeutically relevant knowledge to complex, unanswered and clinically pressing questions, and I am grateful to have been a part of her team during this time; I know that our work will one day lead to personalized treatments for individuals suffering from Cystic Fibrosis disease. In addition, I would like to thank the members of my advisory committee, Drs. John Rubinstein and Régis Pomès, for their expert advice and scientific insight during these past six years. Your significant contributions have allowed me to strive for success in attempting to understand the complex protein structure-function relationship of CFTR. Further, I would like to thank all of the collaborators with whom I have worked with, especially Theo Moraes, Tanja Gonska, Kai Du, Wan Ip, Hong Ouyang and Kethika Kulleperuma. I would also like to thank the place that I called home these past years: the Department of Biochemistry at the University of Toronto, as well as the Department of Molecular Structure and Function at the Hospital for Sick Children. Thank you to Angela Skoutakis for all of her help with administrative work during my studies. I would also like to thank the members of the Bear lab, especially Ling Jun Huan, Drs. Danny Li, Paul Eckford, Mohabir Ramjeesingh, Stan Pasyk, Patrick Kim Chiaw and Leigh Wellhauser for sharing their expertise, and also for their helpful discussions. I would also like to thank Dr. Saumel Ahmadi for his passion and attitude towards science, as well as for his constant assistance and insight into every aspect of my work. He has been a limitless source of ideas, and I really enjoyed working together. I would also like to thank Onofrio Laselva for all of his help with my various projects, as well as for his enthusiasm, ideas and helpful discussions. Also, thank you Michelle Di Paola, Maurita Hung, Stephanie Chin, Randy Kissoon, Ellen Li, Marshall Zhang, Jessica She, Jamie Hu, Wilson Yu and Christina D’Antonio for your helpful insight with research and non-research related subjects alike. It was a pleasure to have worked alongside all of you, thank you for making my experience a v memorable one. Additionally, I would like to say thank you to my family and friends for their support during my time at the University of Toronto. I would especially like to thank my parents, Walter and Luba, as well as my brother, David, and his partner, Diana, for their unconditional love and support. I would also like to thank my ‘new’ family, the Ballas and Skenderis, for their patience, care and support during this stage of my life. Lastly, and most importantly, I would like to express my deepest appreciation and love for my partner in life, Denisa Balla. Your belief in me and my work has provided me with the confidence to strive for success every day. Thank you for being the constant, shining light in my life; I would not be the same man without you by my side. In other words, ti je dielli në mëngjes që ndriçon tokën time. Finally, I must say that I have not done it alone. To quote the great Sir Isaac Newton: “If I have seen further, it is by standing on the shoulders of giants.” vi Table of Contents ABSTRACT ............................................................................................................................ ii STATEMENT OF CO-AUTHORSHIP....................................................................................... iv ACKNOWLEDGEMENTS ........................................................................................................ v TABLE OF CONTENTS ......................................................................................................... vii LIST OF FIGURES ................................................................................................................. xii LIST OF TABLES .................................................................................................................. xvi LIST OF ABBREVIATIONS .................................................................................................. xvii CHAPTER 1: INTRODUCTION 1.1 Acknowledgements ............................................................................................ 1 1.2 ATP-Binding Cassette Transporter Superfamily ................................................. 1 1.3 CFTR and Cystic Fibrosis Disease ....................................................................... 3 1.4 Structural Models of CFTR ................................................................................. 9 1.5 Intra-Molecular Defects Conferred by ΔF508 and Considerations for Less Common Disease-Causing Mutations ...................................................... 13 1.6 Small Molecule Modulators of CFTR and CF Drug Discovery .......................... 16 1.7 Strategies for Personalized CF Medicine ......................................................... 21 1.8 Rationale, Hypotheses, and Objectives ........................................................... 25 CHAPTER 2: MATERIALS AND METHODS 2.1 Generation of mutant CFTR constructs ...........................................................27 2.2 Studies of CFTR protein processing ................................................................. 28 2.3 Studies of CFTR protein degradation ............................................................... 30 2.4 Studies of CFTR protein glycosylation .............................................................. 30 2.5 Preparation of crude membranes, and limited proteolysis of ΔI1234_R1239-CFTR ........................................................................................