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The Significance of the Evolutionary Relationship of Prion Proteins and ZIP Transporters in Health and Disease

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The Significance of the Evolutionary Relationship of Prion Proteins and ZIP Transporters in Health and Disease The Significance of the Evolutionary Relationship of Prion Proteins and ZIP Transporters in Health and Disease by Sepehr Ehsani A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Sepehr Ehsani 2012 The Significance of the Evolutionary Relationship of Prion Proteins and ZIP Transporters in Health and Disease Sepehr Ehsani Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto 2012 Abstract The cellular prion protein (PrPC) is unique amongst mammalian proteins in that it not only has the capacity to aggregate (in the form of scrapie PrP; PrPSc) and cause neuronal degeneration, but can also act as an independent vector for the transmission of disease from one individual to another of the same or, in some instances, other species. Since the discovery of PrPC nearly thirty years ago, two salient questions have remained largely unanswered, namely, (i) what is the normal function of the cellular protein in the central nervous system, and (ii) what is/are the factor(s) involved in the misfolding of PrPC into PrPSc? To shed light on aspects of these questions, we undertook a discovery-based interactome investigation of PrPC in mouse neuroblastoma cells (Chapter 2), and among the candidate interactors, identified two members of the ZIP family of zinc transporters (ZIP6 and ZIP10) as possessing a PrP-like domain. Detailed analyses revealed that the LIV-1 subfamily of ZIP transporters (to which ZIPs 6 and 10 belong) are in fact the evolutionary ancestors of prions (Chapter 3). We were further able to demonstrate that PrPC likely emerged from a ZIP ancestor molecule nearly half-a-billion years ago via a retrotransposition event (Chapter 4). Moreover, biochemical investigations on ZIP10, as a model LIV-1 ZIP transporter, demonstrated that the ectodomain shedding of ZIP10 observed in prion-infected mice resembles a cellular response to transition metal starvation and suggested ii that prion disease in mice might phenocopy a transition metal starvation status (Chapter 5). These studies have opened a new angle to study prion biology in health and disease. Biochemical investigations on other LIV-1 ZIPs and attempts at the structural elucidation of the PrP-like domain of LIV-1 ZIP proteins are ongoing and have not been included in this thesis. iii Acknowledgments I would like to first and foremost thank my thesis supervisor Dr. Gerold Schmitt-Ulms for providing me with a creative, challenging and intellectually-satisfying research project which not only contributed to our collective understanding of aspects of cellular biology, but also had on its horizon the hope of lessening the suffering of those grappling with a dreadful neurodegenerative disease. I would also like to greatly thank the present and past members of my Ph.D. advisory committee, Dr. Rod Bremner, Dr. Sidney E. Croul, Dr. Philip A. Marsden and Dr. Janice Robertson, and the Department of Laboratory Medicine and Pathobiology’s Graduate Coordinator Dr. Harry P. Elsholtz, for their valuable support and guidance. Thank you also to Dr. Glen K. Andrews (University of Kansas Medical Center), Dr. Avi Chakrabartty (University of Toronto) and Dr. John R. Glover (University of Toronto) for evaluating my thesis. During my four years in the graduate program, I was fortunate to be able to collaborate with Joel C. Watts, Hairu Huo and Yu Bai on the prion interactome project, Ashkan Salehzadeh, Hairu Huo and Mohadeseh Mehrabian on the prion/ZIP biochemistry project, Cosmin L. Pocanschi on the ZIP structural project, and Renzhu Tao and Hezhen Ren on the prion/ZIP bioinformatics project. I would like to extend my sincere thanks to them all. Lastly, I would like to thank Robert Strome for teaching me many molecular cloning techniques and Hairu Huo for training me in a number of laboratory techniques. Work on the projects which formed parts of this thesis was funded through support from the Ontario Graduate Scholarship program, the University of Toronto Fellowship program, the Garfield Weston Foundation, the Canadian Institutes of Health Research (MOP-74734) and PrioNet Canada. I would like to dedicate this thesis to my parents. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Appendices ........................................................................................................................ xv Abbreviations ............................................................................................................................... xvi Chapter 1 Prion and ZIP Proteins: An Introduction ........................................................................ 1 1.1 Preamble ............................................................................................................................. 1 1.2 Ancestral ties and diversification ........................................................................................ 2 1.3 Function – novel links to EMT and cancer ......................................................................... 5 1.4 Expression – crosstalk and division of labor .................................................................... 11 1.5 Signaling – from Tinman to Snail and beyond ................................................................. 13 1.6 Post-translational modifications ........................................................................................ 15 1.7 Binding of divalent cations – a common denominator ..................................................... 18 1.8 Conclusions ....................................................................................................................... 21 Chapter 2 Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones ................... 22 2.1 Introduction ....................................................................................................................... 23 2.2 Materials and methods ...................................................................................................... 25 2.2.1 Ethics Statement .................................................................................................... 25 2.2.2 Antibodies ............................................................................................................. 25 2.2.3 Clones ................................................................................................................... 25 v 2.2.4 Cell culture, in vivo crosslinking, inhibitor treatments and cell viability assay ... 26 2.2.5 Affinity purification of bait proteins ..................................................................... 26 2.2.6 Protein reduction, alkylation and trypsinization ................................................... 26 2.2.7 iTRAQ labeling ..................................................................................................... 27 2.2.8 Two-dimensional liquid chromatography ............................................................. 27 2.2.9 Electrospray ionization QqTOF mass spectrometry analysis ............................... 28 2.2.10 Database searches ................................................................................................. 28 2.2.11 Snowdrop lectin affinity purification .................................................................... 29 2.2.12 Cell surface biotinylation ...................................................................................... 29 2.2.13 Proteinase K digestion .......................................................................................... 30 2.3 Results ............................................................................................................................... 30 2.3.1 Large-scale quantitative and comparative interactome investigation of members of the mammalian prion protein family ................................................. 30 2.3.2 Interactions amongst members of the mammalian prion protein family .............. 37 2.3.3 Direct versus indirect PrP interactors ................................................................... 38 2.3.4 PrP forms high-molecular weight complexes with proteins carrying oligomannosidic N-glycans .................................................................................. 39 2.3.5 A subset of cellular P4hb, Pdia3 and calreticulin reside at the cell surface of neuroblastoma cells ............................................................................................... 41 2.3.6 Inhibitors of protein disulfide isomerases increase PrPSc levels in a subset of ScN2a cell clones .................................................................................................. 44 2.4 Discussion ......................................................................................................................... 46 2.5 Conclusion .......................................................................................................................
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