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Chapter One: Introduction Identification of Ryanodine Receptor 1 (RyR1) Interacting Protein Partners Using Liquid Chromatography and Mass Spectrometry by Timothy Ryan A thesis submitted in conformity with the requirements for the degree of Masters of Science Department of Physiology University of Toronto © Copyright by Timothy Ryan 2010 Identification of Ryanodine Receptor 1 (RyR1) Interacting Protein Partners Using Liquid Chromatography and Mass Spectrometry Timothy Ryan Master of Science Department of Physiology University of Toronto 2010 Abstract Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co- localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling. ii Acknowledgments Firstly, I would like to thank my supervisor, Dr. Anthony Gramolini. Since I began my work as a Master’s student, Tony has shown me a great deal of support and provided me with all of the resources necessary to succeed in the lab. I would also like to thank my colleagues in the lab. Parveen, your mentorship and patience from day one in the MacLennan lab, as well as your friendship, have made this process infinitely better. Shaan (styll), Wen-Ping, Thiru, Melissa, and Vijay - thanks for your endless support and all the laughs. Most importantly, I need to thank my family as this process has had its ups and downs. Dad, thanks for the objective advice and constant support. Mom, thanks for the “secret” $20 bills, the soy milk, the cereal, the flattened chickens, the Delissio pizzas, and most importantly, your intense love. Lyndsey, thank you for putting the rye bread in my school bag, it was delicious. Finally, thank you Justin. You have been there (with a bottle of scotch) for the good times, and absorbed the bad with a tolerance and support a kin to that of a true best friend. I would also like to extend my thanks to the Margaret J. Santalo and the Heart and Stroke/Richard Lewar Scholarships for providing me with funding for the course of my Masters degree. iii Table of Contents Abstract.............................................................................................................................. ii Acknowledgments ............................................................................................................ iii List of Common Abbreviations...................................................................................... vii Table of Figures.............................................................................................................. viii List of Tables .................................................................................................................... ix Chapter 1: Introduction ................................................................................................... 1 General Overview ........................................................................................................... 1 I. Skeletal Muscle.................................................................................................... 2 A. Different muscle fiber types.............................................................................. 2 B. Myofibrils ......................................................................................................... 3 C. Sarcoplasmic reticulum and T-tubule system................................................... 4 II. Ryanodine receptors and muscular disorders ...................................................... 5 A. Role of ryanodine receptor in skeletal muscle in SR calcium release .............. 5 B. Structure of ryanodine receptor calcium channel ............................................. 6 C. Modulation of RyR activity .............................................................................. 8 D. Endogenous modulators.................................................................................... 9 E. Exogenous modulators.................................................................................... 10 F. Calcium homeostasis ...................................................................................... 11 III. Neuromuscular disorders ................................................................................... 12 A. General Introduction ....................................................................................... 12 B. Malignant hyperthermia.................................................................................. 14 C. Central core disease ........................................................................................ 14 IV. Protein-protein interactions................................................................................ 15 A. General Introduction ....................................................................................... 15 B. Nickel chelate affinity chromatography.......................................................... 16 C. Ryanodine receptor protein interacting partners............................................. 16 V. Proteomic identification of protein complexes.................................................. 19 A. Proteomic techniques...................................................................................... 19 VI. Statement of Intent............................................................................................. 20 Chapter Two: Materials and Methods.......................................................................... 21 I. Generation of RyR1 cDNA................................................................................ 21 A. Cytoplasmic RyR1 and N-terminally tagged RyR1 D9 fragment .................. 21 B. Chemical Transformation of DH5-α cells ...................................................... 21 C. Amplification and Maxi Preparations of DNA............................................... 21 II. Ni-NTA chromatography purification ............................................................... 22 A. Culturing HEK293 cells.................................................................................. 22 B. Transfection of Cells....................................................................................... 23 C. Harvesting HEK293 cells and preparing protein extract ................................ 23 D. Purification of RyR1 complexes using Ni-NTA resin.................................... 24 iv III. Alternative strategies for purifying functional RyR1 proteins .......................... 26 A. Size exclusion chromatography ...................................................................... 26 B. ELISA Analysis of SEC fractions................................................................... 27 C. Complexiolyte buffer solubilization of FL-RyR1........................................... 27 D. Pull-down of mouse skeletal muscle proteins using bound D9 fragment....... 27 IV. Mass Spectrometry Analysis.............................................................................. 29 A. Protein sample preparation ............................................................................. 29 B. Solid-Phase Extraction of Tryptic Peptides .................................................... 30 C. MS and proteomic analysis of HEK293 cells over-expressing RyR1 ............ 30 D. MS analysis of mouse skeletal muscle lysate on bound D9 fragment............ 31 V. SDS-PAGE and Immunoblot analysis............................................................... 31 A. SDS-PAGE and immunoblot analyses of RyR1 expression........................... 31 B. Immunoblot analysis in HEK293 cells and C2C12 mouse myocytes ............ 32 VI. Subcellular localizations of RyR1 and interactions........................................... 33 A. Plating and fixing slides of HEK-293 cells..................................................... 33 B. Slide Preparations of Fixed Isolated Skeletal Soleus Muscle Fibers.............. 33 C. Co-Immunofluorescent Staining of Fixed Tissue........................................... 33 VII. Calcium transient analysis of RyR1 expressing HEK293 cells with candidate siRNA knockdowns ...................................................................................................... 34 A. Plating slides of HEK293 cells ....................................................................... 34 B. Calcium transient analysis .............................................................................. 34 C. Statistical analysis of data............................................................................... 35 Chapter Three: Results .................................................................................................. 36 I. Purification
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