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Characterization of Cyb5d2 and Its Heme Binding Functions

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Characterization of Cyb5d2 and Its Heme Binding Functions CHARACTERIZATION OF CYB5D2 AND ITS HEME BINDING FUNCTIONS CHARACTERIZATION OF CYB5D2 AND ITS HEME BINDING ASSOCIATED FUNCTIONS By ANTHONY BRUCE, B.Sc., M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Anthony Bruce, September 2013 DOCTOR OF PHILOSOPHY (2013) McMaster University Medical Sciences Hamilton, Ontario, Canada TITLE: Characterization of CYB5D2 and its heme binding associated functions AUTHOR: Anthony Bruce, B.Sc. (University of Western Ontario), M.Sc. (York University) SUPERVISOR: Dr. Damu Tang NUMBER OF PAGES: xxi, 192 ii ABSTRACT Cytochrome b5 heme binding domain 2 (CYB5D2) is a heme binding protein that was initially identified for its ability to attenuate the function of the PTEN tumor suppressor gene. CYB5D2 sustains ectopic PTEN expression in U87 cells, and can also confer survival from serum starvation in NIH3T3 cells. An antibody was generated to the carboxyl terminus of CYB5D2 to detect endogenous protein expression. The highest expression of CYB5D2 protein is in neural cancer cell lines. CYB5D2 is weakly expressed in breast and kidney cancer cell lines, and moderately expressed in prostate cancer cell lines. To investigate the role of the heme binding domain in CYB5D2, a conserved aspartic acid (D86) within this domain was mutated to glycine, and this was characterized as being unable to bind heme. CYB5D2(D86G) displayed a loss of function compared to wild-type CYB5D2. To study the loss of expression of CYB5D2, stable CYB5D2 shRNA was achieved in HeLa and Huh7 cells. While ectopic CYB5D2 inhibited HeLa cell proliferation and growth in soft agar, CYB5D2(D86G) expression and CYB5D2 shRNA increased cell proliferation and soft agar growth. While ectopic CYB5D2 conferred survival from chemotherapeutic drugs in HeLa cells, CYB5D2(D86G) and CYB5D2 shRNA cells were susceptible to drug treatments. CYB5D2 inhibits SREBP signalling, which requires its heme binding ability. Using cyclohexamide treatments, CYB5D2 stabilized ectopic Insig1, while CYB5D2(D86G) destabilized ectopic Insig1. CYB5D2 shRNA reduced endogeneous CYP51A1 (lanosterol demethylase) and Insig1 protein levels, and increased the susceptibility of HeLa cells to mevalonate treatments. Furthermore, CYB5D2 shRNA HeLa cells displayed reduced iii CYP3A4 activity, a cytochrome P450 enzyme involved in drug metabolism. CYB5D2 binds to cytochrome P450 reductase (POR), while CYB5D2(D86G) cannot. CYB5D2 co-immunoprecipitates with endogenous POR under serum-free conditions in HeLa and Huh7 cells, while CYB5D2(D86G) cannot. Collectively, CYB5D2 is a POR interacting protein, which regulates CYP51A1 and CYP3A4 activity. iv FOREWORD This thesis is organized into eight chapters, of which the Results section comprises chapters 3 to 7. Special thanks to the following personnel for their experimental contributions: Dr. Lizhi He, Dr. Fengxiang Wei, Dr. Adrian Rybak, Dr. X. Feng, Dr. Yanyun Xie and Dr. Damu Tang. The cDNA for CYB5D2 was originally cloned by Lizhi He (Chapter 3). Dr. Fengxiang Wei generated GST-CYB5D2 ∆TM constructs and purified the GST-fusion proteins that were subsequently analyzed by absorbance spectrophotometry (Chapter 5). Furthermore, the colorimetric in-gel peroxidase assay used to assess heme binding was also performed by Dr. Fengxiang Wei (Chapter 5). Adrian Rybak contributed and assisted with soft agar assays, xenograft tumor formation assays, and cell viability assays used to evaluate chemotherapeutic drug survival (Chapter 5). Dr. X. Feng performed the Insig stabilization assay by CYB5D2 following cyclohexamide treatment (Chapter 6). CYB5D2 polymorphism mutants (R7P, R7G, Q167K) were generated by Dr. Damu Tang and Dr. Yanyun Xie (Chapter 6). Gross deletion mutants of CYB5D2 (∆TM, ∆CYB5) have been previously published (Xie et al., 2010). v ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Damu Tang, for allowing me to work in his lab. Thank you to Justin He, X. Feng, Cathy Chen, Yanyun Xie and Dr. Fengxiang Wei for all your help. Thanks to Dr. Richard Austin for your interest, your questions and your generosity. Thanks to Steve Colgan and Sana Basserri. I would like to thank my wife, Laurie Lyng, for all your love and support, and the sacrifices that have allowed me to do my Ph.D. studies. I would like to thank my mother, Naty Bruce, for all your love and support. I would like to thank my mother-in-law, Sandra Clegg, for all her help that was required during my studies. Thanks to my daughters, Riley and Avery Bruce. Special thanks goes out to Adrian Rybak for his companionship, his empathy, extensive scientific discussion, his courage and for all his help. vi TABLE OF CONTENTS PAGE CHAPTER 1. INTRODUCTION 1-44 1.0 Heme and Hemoproteins 2 1.1.0 CYB5D2 and PGRMC1 3 1.1.1. Identification of CYB5D2 in a screen for Novel Negative 3 Regulators of the PTEN Tumor Supressor Gene 1.1.2 Structure of CYB5D2 4 1.1.3 CYB5D2 is Heme Binding Protein that Inhibits Neural 5 Differentiation 1.1.4 CYB5D2 Confers Survival from Etoposide 5 1.1.5 Cytochrome B5 Heme Binding Proteins: Progesterone Receptor 6 Membrane Component 1 (PGRMC1) 1.1.6 PGRMC1 (Hpr6) Regulates the Susceptibility of Cancer Cells to 7 Chemotherapeutic Drugs 1.1.7 PGRMC1 is a Sigma 2 Receptor, Connections with Progesterone 8 1.1.8 Yeast Dap1 is a Heme Binding Protein that Regulates Ergosterol 8 Synthesis 1.1.9 PGRMC1 Modulates Cytochrome P450 enzymes 10 vii 1.2.0 SREBP transcription factors and their Regulation 12 1.2.1 PGRMC1 interacts with SCAP and INSIG1 12 1.2.2 Sterol Regulation of the SREBP transcription factors 12 1.2.3 SREBP Activation Regulated by AKT/PKB Signalling 16 1.3.0 Insig interaction with SCAP and HMG CoA reductase: a central role 17 in sterol regulation 1.3.1 HMG CoA reductase 17 1.3.2 Degradation of HMG CoA reductase through Insig1 18 1.3.3 Lanosterol, an intermediate in the synthesis of cholesterol, promotes the Insig-mediated degradation of HMG CoA reductase 19 1.3.4 Gp78 is a membrane ubiquitin ligase, associated with 20 Insig1 (and couples lanosterol-regulated ubiquitination to degradation of HMG CoA reductase) 1.3.5 SCAP and HMG CoA Reductase bind to the same region on Insig1 21 1.3.6 Sterol regulated ubiquitination and degradation of Insig1 by gp78 22 1.4.0 A connection between PGRMC1 and cytochrome P450 reductase (POR) 23 1.4.1 PGRMC1 Heme binding defective mutants 23 1.4.2 Cytochrome P450 enzymes 24 1.4.3 PGRMC1 inhibits the activity of drug-metabolizing cytochrome P450s and binds to cytochrome P450 reductase (POR) 26 1.4.4 Cytochrome P450 enzymes and drug metabolism 27 1.4.5 Roles and involvment of cytochrome P450 enzymes in 28 cholesterol metabolism 1.4.6 Cytochrome P450 reductase (POR) mutations 29 viii 1.4.7 Cytochrome P450 reductase (POR) knockout mice 31 and PORLow mice 1.4.8 Cytochrome P450 reductase (POR) and cholesterol 32 1.4.9 Tissue specific liver knockouts of cytochrome P450 reductase 34 (POR) in mice develop fatty liver 1.5. Hypothesis, Objectives and Summary of thesis work 36 1.5.1 Hypothesis 36 1.5.2 Objectives 37-38 1.5.3 Summary of thesis work 38-44 CHAPTER 2. MATERIALS AND METHODS 45-66 2.1 Cloning of CYB5D2 45 2.2 Plasmids and constructs 45 2.3 Cesium Chloride/Ethidium bromide (CsCl/EthBr) DNA preparation 46 2.4 Generation of a carboxyl terminal CYB5D2 polyclonal antibody 48 2.5 Injection of rabbits and purification of rabbit immunoglobulin 49 (IgG) from serum. 2.6 Calcium Chloride Transfection Method 50 2.7 Generating Stable cell lines of CYB5D2 50 2.8 Generating stable shRNA-mediated CYB5D2 knockdown cells 51 2.9 Construction of CYB5D2 Mutants through site-directed 52 mutagenesis 2.10 Heme binding assays demonstrating D86G is Heme binding 54 defective 2.11 In Gel Peroxidase assay 55 ix 2.12 Oxidation and reduction of CYB5D2 55 2.13 Reporter Assays (Luciferase Assays) 56 2.14 Lysates for Western blot analyses and co-immunoprecipitations 58 2.15 RNA Extractions 61 2.16 Quantitative PCR (qPCR) 62 2.17 Soft agar assays 63 2.18 Tumour xenograft formation 64 2.19 Cell viability assays 64 2.20 Growth curves (Evaluation of cell proliferation) 65 2.21 P450 CYP3A4 Activity assays 65 2.22 Mevalonate treatment 65 2.23 Immunofluorescence 66 2.24 Statistics 66 RESULTS: 67-159 CHAPTER 3. CYB5D2 attenuates PTEN function, 67-81 and confers survival from serum starvation 3.1 CYB5D2 confers survival from PTEN in U87 cells 67 3.2 CYB5D2 confers survival from serum starvation in NIH3T3 cells 73 CHAPTER 4. Generation of a COOH-terminal 82-92 Polyclonal Antibody to CYB5D2 4.1 Generation of a COOH-terminal Polyclonal antibody to CYB5D2 82 x CHAPTER 5. CYB5D2 binds to Heme and a D86G 93-117 Heme binding defective mutant demonstrates loss of functions 5.1 D86 mediates a direct association of heme with CYB5D2 94 5.2 CYB5D2 binds to Heme b, and exists in both oxidized and reduced forms 99 5.3 Heme-binding contributes to CYB5D2-mediated regulation of HeLa cell proliferation and anchorage-independent growth 100 5.4 Heme Binding of CYB5D2 is required for survival from chemotherapeutic compounds 109 5.5 Generation and characterizatin of EV, CYB5D2, D86G, shRNA control and shRNA CYB5D2 stable Huh7 cell lines: Heme binding is required for CYB5D2 to regulate proliferation in Huh7 cells. 114 CHAPTER 6. Heme binding of CYB5D2 is required for 118-139 inhibition of SREBP signalling 6.1 CYB5D2 Co-Immunoprecipitates with Insig1 125 6.2 CYB5D2 Stabilizes Ectopic Insig1 127 6.3 The transmembrane domain and heme binding are required 131 for CYB5D2 to inhibit SREBP signalling 6.4 The D86G CYB5D2 destabilizes ectopic Insig1 135 CHAPTER 7.
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