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Structural Studies of the Mechanism by Which Bcl-2 and Beclin STRUCTURAL STUDIES OF THE MECHANISM BY WHICH BCL-2 AND BECLIN PROTEINS REGULATE AUTOPHAGY AND APOPTOSIS A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Minfei Su In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Chemistry and Biochemistry July 2016 Fargo, North Dakota North Dakota State University Graduate School Title STRUCTURAL STUDIES OF THE MECHANISM BY WHICH BCL-2 AND BECLIN PROTEINS REGULATE AUTOPHAGY AND APOPTOSIS By Minfei Su The Supervisory Committee certifies that this disquisition complies with North Dakota State University‟s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Sangita Sinha Chair Dr. Mukund Sibi Dr. Bin Guo Dr. Glenn Dorsam Approved: July 6, 2016 Dr. Gregory Cook Date Department Chair ABSTRACT Autophagy and apoptosis are catabolic pathways essential for homeostasis in all eukaryotes. While autophagy usually promotes cell-survival by enabling degradation and recycling of damaged macromolecules, apoptosis is the canonical programmed cell death pathway. Dysfunction of autophagy and apoptosis is implicated in diseases like cancers, cardiac disease, and infectious disease. Beclin homologs are key for autophagy as they are core components of class III phosphatidylinositol 3-kinase autophagosome nucleation and maturation complexes. Anti-apoptotic Bcl-2s inhibit apoptosis through binding and antagonizing pro- apoptotic Bcl-2s. Anti-apoptotic Bcl-2s also down-regulate autophagy by binding to the Bcl-2 homology domain 3 (BH3D) of Beclin homologs, thereby enabling crosstalk between apoptosis and autophagy. The central goal of my doctoral research is to understand the structure-based mechanism of selected Bcl-2s and Beclin homologs in the regulation of autophagy and apoptosis. -Herpesviruses are common human pathogens that encode viral Bcl-2s to facilitate viral reactivation and oncogenic transformation. M11, a viral Bcl-2, and Bcl-XL, a cellular Bcl-2, bind with comparable affinities to the Beclin 1 BH3D. By assessing the impact of different Beclin 1 BH3D mutations on binding to M11 and Bcl-XL, we developed a cell-permeable inhibitory peptide that targets M11, but not Bcl-XL. The mechanism by which this peptide specifically binds to M11 was elucidated by determining the X-ray crystal structure of the peptide:M11 complex. Our attempts to investigate the role of other Bcl-2s in these pathways were unsuccessful. In one project, we were unable to express and purify BALF1, another viral Bcl-2. In another, no interaction was detected between purified samples of the anti-apoptotic Bcl-2 homolog Mcl-1, and the autophagy protein Atg12, that had previously been shown to bind. iii We also delineated the domain architecture of Beclin 2, a novel Beclin homolog and attempted to express and purify different Beclin 2 constructs for structural studies. We successfully purified and solved the X-ray crystal structure of the Beclin 2 coiled-coil domain (CCD), showing it is a curved, anti-parallel, meta-stable coiled-coil homodimer with seven pairs of non-ideal packing residues. In general, mutating the non-ideal packing residues to leucines enhanced Beclin 2 CCD homodimerization, but also weakened its binding to Atg14 CCD. iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor Dr. Sangita Sinha for all her guidance, support, and encouragement throughout my graduate studies. Without her, this work would not have been possible. I would also like to thank my committee members, Dr. Mukund Sibi, Dr. Bin Guo and Dr. Glenn Dorsam for their advice and direction. I would like to thank our collaborator, Dr. Christopher Colbert here at North Dakota State Univerity, not only for his collaboration, but also for all his continuous advice and support. I would also like to thank our other collaborator Dr. Beth Levine group at University of Texas Southwestern Medical Center, Dr. David Neau at NE-CAT, APS, Argonne, Dr. Ruslan Sanishvili at GMCA, APS, Argonne, and Dr. Srinivas Chakravarthy st Bio-CAT, APS, Argonne, for their collaboration. Additionally, I would like to thank all the members in the Sinha group, including Yang Mei, Yue Li, Karen Glover, Shreya Mukhopadhyay, Srinivas Dasanna, Marion Okondo, Tanner Huebner, Vishnuveni Leelaruban, and Samuel Wyatt, and Colbert group, including Jaime Jensen Shane Wyborny and Benjamin LeVahn, who made my graduate experience both enjoyable and productive through daily advice, assistance, and relaxation. This research was supported and funded by NIH grant R21 AI078198, NIH grant R03 NS090939, NSF grant MCB 1413525, NSF grant HRD-0811239, and P30 GM103332-01 to Dr. Sangita Sinha, and North Dakota EPSCoR doctoral dissertation award for Minfei Su and Dr. Sangita Sinha. v DEDICATION This dissertation is dedicated to my parents, for their endless love, understanding, support, encouragement and inspiration. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................................ v DEDICATION ............................................................................................................................... vi LIST OF TABLES ....................................................................................................................... xiii LIST OF FIGURES ..................................................................................................................... xiv LIST OF ABBREVIATIONS ..................................................................................................... xvii CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1. Introduction to autophagy .................................................................................................... 1 1.2. Introduction to apoptosis ...................................................................................................... 3 1.3. The molecular crosstalk between autophagy and apoptosis ................................................. 5 1.3.1. Bcl-2 proteins ................................................................................................................ 5 1.3.2. The Beclin 1–Bcl-2 interaction ..................................................................................... 8 1.3.3. Caspase-mediated Beclin 1 cleavage............................................................................. 9 1.3.4. BIM and its role in different autophagy stages ........................................................... 10 1.3.5. Interaction of UVRAG with BAX ............................................................................... 11 1.3.6. Interaction of UVRAG with Bif-1 ............................................................................... 11 1.3.7. Atg12–Atg3 conjugation ............................................................................................. 12 1.3.8. Atg5–FADD interaction .............................................................................................. 12 1.3.9. Calpain-mediated Atg5 cleavage ................................................................................. 13 1.3.10. p53: A master regulator of autophagy and apoptosis ................................................ 13 1.3.11. The regulation of autophagy by anti-apoptotic viral Bcl-2 homologs ...................... 15 1.4. Overview of methods used in this research ........................................................................ 16 1.4.1. Protein X-ray crystallography ..................................................................................... 16 1.4.2. Small-angle X-ray scattering (SAXS) ......................................................................... 22 vii 1.4.3. Isothermal titration calorimetry(ITC) .......................................................................... 27 1.4.4. Circular dichroism (CD) .............................................................................................. 30 1.5. Specific aims of this study ................................................................................................. 32 CHAPTER 2. DEVELOPMENT OF A PEPTIDE INHIBITOR THAT SPECIFICALLY BLOCKS THE DOWN-REGULATION OF AUTOPHAGY BY VIRAL BCL-2 OF MURINE γ-HERPESVIRUS 68 ................................................................................................... 34 2.1. Introduction ........................................................................................................................ 34 2.1.1. γHVs encode Bcl-2 homologs ..................................................................................... 34 2.1.2. Cellular and γHVBcl-2 homologs ............................................................................... 35 2.1.3. Beclin 1 BH3D ............................................................................................................ 41 2.1.4. NOXA BH3D .............................................................................................................. 46 2.1.5. In this study ................................................................................................................. 47 2.2. Materials and methods ......................................................................................................
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