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Identification of the Binding Partners for Hspb2 and Cryab Reveals Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal- induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2- binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function. Keywords: small heat shock proteins (sHSP), HspB2 (MKBP), HspB5 (CryAB), R120G, A177P, molecular chaperones, myocardial maintenance strategies, mitochondria, proteostasis TABLE OF CONTENTS TITLE PAGE ..…………………………………………………………………………………….i ABSTRACT .................................................................................................................................... ii TABLE OF CONTENTS ............................................................................................................... iii LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ........................................................................................................................ 1 SPECIFIC AIMS ............................................................................................................................ 2 CHAPTER ONE: HspB2 Responds to Cardiac Stress ................................................................... 5 INTRODUCTION ....................................................................................................................... 5 SMALL HEAT SHOCK PROTEINS PLAY CONSERVATIONAL ROLES IN CELL SURVIVAL ................................................................................................................................. 6 THE SMALL HEAT SHOCK PROTEIN HSPB2 ..................................................................... 8 Genetic Features and Expression Patterns ............................................................................... 8 Cellular Localization ............................................................................................................. 10 Molecular Properties of HspB2 ............................................................................................. 12 Chaperone Activity and Other Protein Interactions of HspB2 .............................................. 14 PHENOTYPES ASSOCIATED WITH HSPB2 ....................................................................... 15 Knockout Effects Under Normal Physiological Conditions.................................................. 15 Knockout Effects Upon Ischemia/Reperfusion (I/R) Stress Conditions ............................... 16 Knockout Effects Upon Myocardial Mechanical or Chronic Pressure-Overload Stress Conditions .............................................................................................................................. 17 iii Anti-Apoptotic Properties...................................................................................................... 18 CONCLUSION ......................................................................................................................... 21 CHAPTER TWO: Identification of the HspB2 Cardiac Interactome Reveals Unique Mitochondrial and Muscle Clients Involved in Myopathies and Neurodegenerative Disease ..... 23 ABSTRACT .............................................................................................................................. 23 INTRODUCTION ..................................................................................................................... 23 METHODS................................................................................................................................ 25 Yeast Two-Hybrid (Y2H) Screen and HspB2 Dependency Tests ........................................ 25 HSPB2cTG Mouse Line ........................................................................................................ 25 Mitochondrial Coaffinity Proteomics Experiments ............................................................... 26 Bioinformatics/Network Analysis ......................................................................................... 26 HspB2 and CryAB Binding Partner Comparison .................................................................. 27 RESULTS.................................................................................................................................. 27 An Unbiased Yeast Two-hybrid (Y2H) Screen for HspB2 Substrates Identifies Novel Cardiac Clients ...................................................................................................................... 27 Validating the Y2H HspB2 Interactome Through Coaffinity Proteomics. ........................... 34 The Y2H and Copurificaton HspB2 Mitochondrial Interactomes Display 80% Overlap. .... 39 Network Analysis of the HspB2 Interactome Reveals Links to Myopathies and Neurodegenerative Disease. .................................................................................................. 43 Comparison of HspB2 and CryAB Binding Partners Reveals Remarkable HspB2 Specificity. ............................................................................................................................. 44 iv CHAPTER THREE: Identification of WTCryAB and R120GCryAB Clientele Reveals R120G to Have Hyperactive Binding Activity and Aggregation with Clientele .......................................... 55 ABSTRACT .............................................................................................................................. 55 INTRODUCTION ..................................................................................................................... 55 METHODS................................................................................................................................ 57 Yeast Two-Hybrid (Y2H) Screen .......................................................................................... 57 R120GCryAB and WTCryAB Binding Partner Comparison ...............................................
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