Western University Scholarship@Western Electronic Thesis and Dissertation Repository 6-24-2019 10:30 AM Apolipoprotein(a) Secretion is Modulated by Sortilin, Proprotein Convertase Subtilisin/Kexin Type 9, and Microsomal Triglyceride Transfer Protein Justin Clark The University of Western Ontario Supervisor Koschinsky, Marlys L. Robarts Research Institute Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Justin Clark 2019 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cardiovascular Diseases Commons Recommended Citation Clark, Justin, "Apolipoprotein(a) Secretion is Modulated by Sortilin, Proprotein Convertase Subtilisin/Kexin Type 9, and Microsomal Triglyceride Transfer Protein" (2019). Electronic Thesis and Dissertation Repository. 6310. https://ir.lib.uwo.ca/etd/6310 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Elevated plasma lipoprotein(a) (Lp(a)) levels are a causal risk factor for cardiovascular disease (CVD), but development of specific Lp(a) lowering therapeutics has been hindered by insufficient understanding of Lp(a) biology. For example, the location of the noncovalent interaction that precedes the extracellular disulfide linkage between apolipoprotein(a) (apo(a)) and apolipoprotein B-100 (apoB-100) in Lp(a) biosynthesis is unclear. In this study we modulated known intracellular regulators of apoB-100 production and then assessed apo(a) secretion from human HepG2 cells expressing 17-kringle (17K) apo(a) isoform variants using pulse-chase analysis. Treating 17K-expressing HepG2 cells with proprotein convertase subtilisin-kexin type 9 (PCSK9) significantly increased apo(a) secretion. Treating the same cell line with Lomitapide, a microsomal triglyceride transfer protein (MTP) inhibitor, significantly decreased apo(a) secretion. Overexpression of human sortilin variants (F404Y and K302E) significantly increased apo(a) secretion relative to wild-type. Our findings suggest a role for sortilin, PCSK9, and MTP in modulating Lp(a) levels through effects on apo(a) secretion, possibly through impacting the intracellular bioavailability of apoB-100. Keywords Cardiovascular Disease, Lipoprotein(a), apolipoprotein(a), apolipoprotein B-100, sortilin, proprotein convertase subtilisin-kexin type 9, Lomitapide, microsomal triglyceride transfer protein ii Lay Summary Lipoprotein(a) (Lp(a)) is a circulating lipoprotein that contributes to the development of cardiovascular disease. The structure of lipoprotein(a) consists of a low-density lipoprotein- like core that is covalently attached to apolipoprotein(a). Unfortunately, no pharmacological therapies designed to specifically lower Lp(a) currently exist, which reflects a lack of fundamental understanding of the mechanisms regulating Lp(a) production and catabolism. In this study, we modulated known regulators of apoB-100 secretion and subsequently analyzed apo(a) secretion to determine if a non-covalent interaction exists between apo(a) and apoB- 100 in Lp(a) production. iii Co-Authorship Statement All of the data in this thesis was collected by Justin Clark. Dr. Michael Boffa and Dr. Marlys Koschinsky contributed to the general supervision of work, including the generation of appropriate experimental designs and editing of this thesis. iv Acknowledgments Firstly, I would like to thank Dr. Marlys Koschinsky for giving me the opportunity to pursue graduate studies in her lab. Your guidance and expertise were integral to the development of my skills as a scientist. I would also like to thank Dr. Michael Boffa for his contributions to my project. Your advice and assistance in troubleshooting helped propel my project forward. Secondly, I would like to thank my advisory committee members, Dr. Michael Boffa, Dr. Nica Borradaile, and Dr. Timothy Regnault. Thank you all for your support and insight. Your feedback was instrumental to the progression of my project. I would also like to thank the members of Koschinsky lab. Thank you to Dr. Amer Youssef for working closely with me and allowing me to learn from you. Thank you to Julia St. John for helping me obtain all of the resources necessary to complete my experiments, and for everything else that you do. Your hard work and help in the lab is appreciated by all of us. Thank you to Matthew Borrelli for your support and all of the laughs over the past two years. Finally, I would like to thank all of my family and friends for their continued encouragement and support. Specifically, I would like to thank my parents, Robert and Jennifer, for always believing in me. v Abstract ........................................................................................................................... ii Lay Summary .................................................................................................................. iii Co-Authorship Statement ................................................................................................ iv Acknowledgments ............................................................................................................ v List of Tables ................................................................................................................... ix List of Figures ................................................................................................................... x List of Abbreviations ........................................................................................................ xi Chapter 1 ......................................................................................................................... 1 1 Introduction .............................................................................................................. 1 1.1 Cardiovascular Disease: Significance & Pathogenesis .................................................... 1 1.2 Lipoprotein(a) and Apolipoprotein(a) ........................................................................... 3 1.2.1 Beyond LDL-cholesterol .................................................................................................................. 3 1.2.2 Lp(a) as a Causal Risk Factor for CVD .............................................................................................. 3 1.2.3 Structure of Lp(a) and Apo(a) ......................................................................................................... 5 1.2.4 Proposed Mechanisms of Lp(a) Pathophysiology ........................................................................... 9 1.2.5 Determinants of Plasma Lp(a) Concentrations ............................................................................. 14 1.2.6 Lp(a) Metabolism .......................................................................................................................... 16 1.3 Proprotein Convertase Subtilisin/Kexin Type 9 ........................................................... 23 1.3.1 Discovery & Defining Features ..................................................................................................... 23 1.3.2 The LDL-Receptor & PCSK9 ........................................................................................................... 23 1.3.3 PCSK9 Inhibitors ........................................................................................................................... 25 1.3.4 PCSK9 & Lp(a) Metabolism ........................................................................................................... 26 1.4 Microsomal Triglyceride Transfer Protein ................................................................... 27 1.4.1 Structure & Function .................................................................................................................... 27 1.4.2 MTP & ApoB-containing Lipoprotein Assembly ........................................................................... 28 1.4.3 Implications with Lp(a) ................................................................................................................. 29 1.5 Sortilin ....................................................................................................................... 30 1.5.1 Genome-Wide Association Studies .............................................................................................. 30 1.5.2 Structure & Function .................................................................................................................... 31 1.5.3 Sortilin’s Enigmatic Role in LDL Metabolism ................................................................................ 35 vi 1.5.4 Sortilin & Lp(a) .............................................................................................................................. 37 1.6 Rationale, Objectives & Hypotheses ........................................................................... 39 Chapter 2 ....................................................................................................................... 41 2 Methods ................................................................................................................. 41 2.1 Cell Culture ................................................................................................................ 41