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Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay

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Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay Copyright by John Francis Anderson 2011 THE ROLE OF SACSIN AS A MOLECULAR CHAPERONE by John Francis Anderson, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas Medical Branch May 2011 Dedication To my wife, Erika R. Anderson. Acknowledgements I am indebted to my mentor, Dr. José M. Barral for his investment in my education. Dr. Barral taught me that curiosity drives scientific investigation and rigorous experiments derive new knowledge. This combination of values is rare to find in a mentor and I am extremely grateful for my experience in his lab. I would like to acknowledge Jason J. Chandler and Paige Spencer for technical assistance. I am especially grateful to Dr. Efrain Siller for daily assistance and discussions on all aspects of this project. I would like to thank Dr. Christian Kaiser providing me a practical education on the design and performance biochemical experiments. I acknowledge Dr. Bernard Brais for generously sharing his time and resources; he provided the rare opportunity to visit an ARSACS patient in her home as well as supplied us with SACS knockout mouse brains. My committee members, Dr. Henry F. Epstein, Dr. Andres F. Oberhauser, Dr. George R Jackson, and Dr. Robert O. Fox and Dr. Darren F. Boehning provided invaluable guidance throughout this work for which I am very grateful. I would like to especially thank Dr. Darren F. Boehning and Dr. Henry F. Epstein for being involved in the details of my project from the beginning of my education at UTMB, through sharing reagents and expertise as well as serving on my committee. Financial support for this study was supplied by the March of Dimes Foundation, the Pew Foundation and the Jean C. and William D. Willis foundation. I am especially grateful to Dr. William D. Willis and others who contributed to the Jean C. and William D. Willis foundation. v THE ROLE OF SACSIN AS A MOLECULAR CHAPERONE Publication No._____________ John Francis Anderson, Ph.D. The University of Texas Medical Branch, 2011 Supervisor: José M. Barral The central pathological finding in neurodegenerative disease is loss of neurons. In many disorders, this loss of neurons appears to be related to protein misfolding, comprising a large public health burden. For example, the two most common neurodegenerative diseases, Alzheimer’s and Parkinson’s diseases, possess protein misfolding as a core component of their pathology. In order to properly fold, many proteins require the assistance of molecular chaperones. While substantial gains in our knowledge of the function of general chaperones have been made in the last two decades, the role of molecular chaperones in brain-specific processes is not clearly defined. In this study, we examined the function of the protein sacsin, mutated in autosomal recessive spastic ataxia of Charlevoix-Saguenay. Pathologically, these patients demonstrate loss of neurons in the cerebellum and cervical spinal cord along with inclusions reminiscent of misfolded proteins in the remaining neurons. Sacsin contains regions of similarity to both molecular chaperones and co-chaperones. Thus, this disease may represent a useful model to study the role of molecular chaperones in neurodegenerative disease. We vi performed bioinformatics and biochemical investigations to determine the function of sacsin. We found that this protein contains a novel supra-domain present three times. We show that this domain is both ATPase active and contains molecular chaperone activity. Additionally, we determined that a region of sacsin possesses co-chaperone activity. Thus, sacsin is a novel molecular chaperone, with built-in co-chaperone modules, directly involved in a neurodegenerative disease. vii Table of Contents List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Abbreviations .....................................................................................................................xv SECTION 1: INTRODUCTION ..............................................................................................1 Chapter 1: Protein Biogenesis and Folding .........................................................................1 A. Protein Biogenesis and Stability ............................................................................1 B. Protein Folding and Neurodegeneration ..............................................................18 C. The Role of Molecular Chaperones in Protein Folding .......................................24 Chapter 2: Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay .......................50 A. Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay ...........................50 B. The Sacsin Protein ................................................................................................61 SECTION 2: RESULTS .......................................................................................................71 Chapter 3: Sacsin Contains a Novel Supra-Domain with Similarity to a Region of Hsp90 ........................................................................................................................71 A. There Is a Repeating Supra-Domain in Sacsin with Similarity to Hsp90: SRR ..72 B. The Phylogeny of SRR Domains Reveals Multiple Instances of Convergent Evolution ..........................................................................................................83 C. Conclusions ..........................................................................................................89 Chapter 4: The SRR Domain Is ATPase Active ................................................................91 A. Production of a Soluble Fragment of Sacsin ........................................................91 B. Purification of a Soluble Fragment of Sacsin.......................................................98 C. Biochemical Properties of Sacsin .......................................................................105 Chapter 5: Sacsin Is Both a Chaperone and a Co-Chaperone ..........................................112 A. Sacsin Is a Chaperone ........................................................................................112 B. Sacsin Is a Co-chaperone ...................................................................................127 C. Conclusions ........................................................................................................131 viii Chapter 6: Potential Client Proteins of Sacsin .................................................................133 A. Sacsin Interacts with MAP1A ............................................................................133 B. Analysis of Sacsin Knockout Mouse Brain........................................................138 C. Conclusions ........................................................................................................142 SECTION 3: DISCUSSION ................................................................................................143 Chapter 7: The SRR Supra-Domain ................................................................................143 Chapter 8: The Protein Folding Activities of Sacsin .......................................................149 Chapter 9: Conclusions and Perspectives ........................................................................154 Conclusions .............................................................................................................154 Perspectives.............................................................................................................154 SECTION 4: MATERIALS AND METHODS ......................................................................156 Chapter 10: Methods ........................................................................................................156 A. Bioinformatics Analysis .....................................................................................156 B. Buffer Compositions ..........................................................................................156 C. Cloning ...............................................................................................................159 D. Purification of Proteins ......................................................................................160 E. Cell Culture ........................................................................................................165 F. SDS-PAGE .........................................................................................................165 G. Western Blots .....................................................................................................166 H. Fluorescence Spectrocopy ..................................................................................166 I. Immunoprecipitations ..........................................................................................167 J. Luciferase Refoldings..........................................................................................167 K. Dot Blot Analysis ...............................................................................................168 L. Immunofluorescence ..........................................................................................168
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