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The Direct and Functional Interaction of Tubulin with Transient Receptor Potential Melastatin 2

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The Direct and Functional Interaction of Tubulin with Transient Receptor Potential Melastatin 2 The Direct and Functional Interaction of Tubulin With Transient Receptor Potential Melastatin 2 by Colin Elliott Seepersad A thesis submitted in conformity with the requirements for the degree of Masters of Science Cell & Systems Biology University of Toronto © Copyright by Colin Elliott Seepersad 2011 The Direct and Functional Interaction of Tubulin With Transient Receptor Potential Melastatin 2 Colin Elliott Seepersad Masters of Science Cell & Systems Biology University of Toronto 2011 Abstract Transient Receptor Potential Melastatin 2 (TRPM2) is a widely expressed, non-selective cationic channel with implicated roles in cell death, chemokine production and oxidative stress. This study characterizes a novel interactor of TRPM2. Using fusion proteins comprised of the TRPM2 C-terminus we established that tubulin interacted directly with the predicted C-terminal coiled-coil domain of the channel. In vitro studies revealed increased interaction between tubulin and TRPM2 during LPS-induced macrophage activation and taxol-induced microtubule stabilization. We propose that the stabilization of microtubules in activated macrophages enhances the interaction of tubulin with TRPM2 resulting in the gating and/or localization of the channel resulting in a contribution to increased intracellular calcium and downstream production of chemokines. ii Acknowledgments I would like to thank my supervisor Dr. Michelle Aarts for providing me with the opportunity to pursuit a Master’s of Science Degree at the University of Toronto. Since my days as an undergraduate thesis student, Michelle has provided me with the knowledge, tools and environment to succeed. I am very grateful for the experience and will move forward a more rounded and mature student. Special thanks to my committee members, Dr. Mauricio Terebiznik, and Dr. Dinesh Christendat who provided the insight and direction for my progress. I would also like to acknowledge NSERC and the University of Toronto Fellowship for providing the funding necessary for my studies. I extend my gratitude and thanks to the many friends I’ve made along the way. Past and present members of the Aarts Lab, you have provided the support, advice and humor needed to keep me going; Danny Chan, Kevin Sam, Russell Bent, Melanie Ratnam, Darren Gigliozzi, Naghmeh Lesani and Jonathan Chan – Thank you. I am most gracious for the external support and the endless amount of advice, expertise and RAW cells – thank you to all the members of the Harrison and Terebiznik Labs. Thanks to Chris Yong-Kee, Sherri Thiele, Ari Chow and Sam Khalouei. Extended thanks to Dr. Shelley Brunt and Dr. Mauricio Terebiznik for your invaluable and warm advice, support and expertise. Lastly I would like to thank my friends and family for the love, encouragement and motivation. iii Table of Contents Acknowledgments...................................................................................................................................................iii Table of Contents.....................................................................................................................................................iv List of Tables.............................................................................................................................................................vii List of Figures......................................................................................................................................................... viii List of Appendices ...................................................................................................................................................ix List of Abbreviations............................................................................................................................................... x Chapter 1......................................................................................................................................................................1 1 Introduction ........................................................................................................................................................1 1.1 Transient Receptor Potential Channels..........................................................................................................1 1.2 The TRPM Subfamily...............................................................................................................................................5 1.2.1 TRPM1 ..................................................................................................................................................................5 1.2.2 TRPM3 ..................................................................................................................................................................6 1.2.3 TRPM4 & TRPM5 .............................................................................................................................................6 1.2.4 TRPM6 & TRPM7 .............................................................................................................................................7 1.2.5 TRPM8 ..................................................................................................................................................................7 1.3 TRPM2...........................................................................................................................................................................7 1.4 TRPM2 Modes of Activation ............................................................................................................................. 13 1.5 The Pharmacology of TRPM2........................................................................................................................... 16 1.6 The Physiological and Pathological Roles of TRPM2............................................................................. 16 1.7 Hypothesis and Objectives ................................................................................................................................ 18 Chapter 2................................................................................................................................................................... 20 2 Materials and Methods................................................................................................................................ 20 2.1 Cloning ....................................................................................................................................................................... 20 2.1.1 The Generation of GST-Fusion Constructs......................................................................................... 20 2.2 Protein Pull-Down and Tubulin Interaction Assay................................................................................. 23 2.2.1 Protein purification...................................................................................................................................... 23 iv 2.2.2 Mouse Brain Homogenization................................................................................................................. 24 2.2.3 Protein Pull-Down Assay........................................................................................................................... 24 2.2.4 Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).............. 25 2.2.5 Tubulin Binding Assay................................................................................................................................ 25 2.3 SDS-Polyacrylamide Gel Electrophoresis ................................................................................................... 25 2.4 Western Blotting.................................................................................................................................................... 25 2.5 Cell Culture............................................................................................................................................................... 26 2.6 Immunocytochemistry........................................................................................................................................ 26 Chapter 3................................................................................................................................................................... 27 3 Results ................................................................................................................................................................ 27 3.1 Identifying TRPM2 C-Terminal Protein Interactions ............................................................................ 27 3.1.1 Generation of Full-Length GST-TRPM2 C- and N-Terminus Fusion Constructs................ 27 3.1.2 Expression of GST-TRPM2 C- and N-Terminus Fusion Constructs in BL-21 DES E. coli 33 3.1.3 Identification of TRPM2 C-Terminus Protein Interactors Through Protein-Pull Down Assays and Mass Spectrometry.............................................................................................................................. 34 3.2 Investigating Tubulin’s Interaction With the Full-Length TRPM2 C-Terminus......................... 34 3.2.1 Tubulin Binds Directly to the C-Terminus of TRPM2 ................................................................... 34 3.3 Investigating the Binding Location of Tubulin Along the C-Terminus of TRPM2..................... 41 3.3.1 Generation
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