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GLUTATHIONE TRANSFERASE N STRUCTURE and GENE REGULATION

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GLUTATHIONE TRANSFERASE N STRUCTURE and GENE REGULATION GLUTATHIONE TRANSFERASE n STRUCTURE AND GENE REGULATION By Chu-Lin Xia This report is in part-fulfillment of the requirements for the degree of doctor of philosophy in the University of London University College, University of London January 1992 ProQuest Number: 10609162 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609162 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT In the early stage of this research, amino acid residues that are essential for the activity of human pi class glutathione transferase (GST n) were identified using chemical modification with group-specific reagents. Protection from inactivation by substrates, substrate analogues and inhibitors was used as the criterion for active site specificity. The results suggested the apparent involvement of one cysteine (Cys), one lysine (Lys), one arginine (Arg), one histidine (His) or tyrosine (Tyr) or both, one aspartate (Asp) or glutamate (Glu) and tryptophan (Trp) residues in the glutathione binding site (G-site) of GSTtc. It was concluded that without the knowledge of the three-dimensional structure of GST 7i, further work in this area would not be profitable and therefore, attention was turned to the regulation of GST n gene expression. Recently, the three-dimensional structure of porcine G ST pi was solved and interpretation of these results in terms of the structure of the porcine GST pi by computerized modelling revealed that Tyr7, Arg13, Trp38, Lys44 and Asp98 were associated with the G-site. The roles of Cys47 and His71 in the active site are not clear. Studies of the regulation of GST n gene involved the fusion of fragments of both the GST n gene and 13.5 kb of its 5’ flanking region to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, EJ and MCF7) demonstrated that the consensus AP1 binding site, located between nucleotides -58 to -65, was essential for the basal level promoter activity of the gene. A positive cis- acting DNA element between nticleotides +8 to +72 was also identified which could be bound by protein(s) both in vivo and in vitro. This element is part of the promoter since it functions in an orientation and position dependent manner. No other regulatory activity was identified within the 17 kb sequence analyzed. Regulation by both retinoic acid and insulin was observed, but the mechanisms of the regulation remain to be elucidated. 2 ACKNOWLEDGEMENTS I would like to thank my supervisor Professor Brian Ketterer, my advisor Dr. Elizabeth Shephard and Professor Peter Campbell for their support and encouragement in this project. I am very grateful to my fellow members of the Cancer Research Campaign Molecular Toxicology Research Group: Drs. John Taylor, David Meyer, Ian Cowell, Sally Pemble, Kathy Dixon, Sharon Spencer and Brian Coles, Misses Kim Gilmore and Gillian Fraser and Mr. Jonathan Harris, for their friendship, advice and support. In particular, I would like to thank Dr. John Taylor and Dr. David Meyer for their expert guidance on my work on the gene regulation of GST n and the structure of the active site of GST n respectively. I would also like to thank Miss Lucia Christodoulides, the laboratory administrator, for many kindnesses and acts of help. Finally, I thank my husband, Dr. Jiahua Wang, for his unfailing encouragement and support and for his help in preparation of this manuscript. The studies reported here were supported by the Frances and Augustus Newman Foundation, the Cancer Research Campaign, the Sino-British Friendship Foundation in collaboration with the British Council and the European Molecular Biology Organization. 3 LIST OF CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 3 LIST OF CONTENTS 4 LIST OF FIGURES 11 LIST OF TABLES 14 LIST OF ABBREVIATIONS 15 PREFACE 18 CHAPTER I INTRODUCTION 20 1.1 Drug metabolism, glutathione, glutathione 21 conjugation and glutathione transferase 1.1.1 Drug metabolism 21 1.1.2 Biosynthesis and metabolism of glutathione and 23 glutathione conjugates 1.1.3 Multifunctional nature of GSTs 25 1.2 Classification of GSTs 29 1.2.1 Purification and identification of GSTs 29 1.2.2 Classification and nomenclature of GSTs 30 1.3 Protein structure and evolutionary relationships 31 of GSTs 1.3.1 Primary structure of GSTs and evolutionary 31 relationships 1.3.2 Secondary, tertiary and quaternary structure 38 1.4 Structure, organization and chromosomal 40 location of GST genes 1.5 Catalytic mechanism and structure of the 42 active site 1.5.1 The kinetic mechanism of GSTs 42 1.5.2 The catalytic mechanism of GSTs 42 1.5.3 The active site of GSTs 1.6 GSTs expression in rat and human 45 1.6.1 Tissue distribution of GSTs in rat and man 45 1.6.2 Developmental change of GSTs expression 47 4 Content 1.7 Modulation of GST expression 47 1.7.1 Post-translational modification 47 1.7.2 Hormonal regulation of GST expression 48 1.7.3 Chemical induction of GSTs 49 1.8 Expression of Pi class GSTs in carcinogenesis 49 and drug resistance 1.8.1 GST 7-7 and carcinogenesis 49 1.8.2 GST n and human tumours 51 1.8.3 GST n and drug resistance 53 1.9 Control of the expression of GST genes 56 1.9.1 Control of the expression of mouse GST 56 subunit Ya gene 1.9.2 Control of the expression of rat GST 57 subunit 1b gene 1.9.3 Control of the expression of rat GST 60 subunit 7 gene 1.9.4 Control of the expression of human GST n gene 63 1.10 Aim of this thesis 63 CHAPTER II MATERIALS AND METHODS 65 Foreword 66 PART I 66 2.1.1 Chemicals 66 2.1.2 Protein determination 67 2.1.3 Enzyme activity assay 67 2.1.4 Purification of GST n from human placenta 67 2.1.5 Modification by DTNB 68 2.1.5.1 Reaction of GST n with DTNB 68 2.1.5.2 Determination of the rate constant of 68 DTNB modification 2.1.5.3 Effect of GS-OCT on DTNB modification 68 2.1.6 Modification by NEMI 69 2.1.6.1 Kinetics of NEMI inactivation of GST n 69 2.1.6.2 Effects of GS-OCT, CDNB and bilirubin 69 5 on NEMI inactivation of GST n Modification of GST n by TNBS 69 Modification by TNBS 69 Effects of GS-OCT, bilirubin and CDNB 70 on TNBS inactivation of GST n Modification of GST n by DiAC 70 Kinetics of DiAC modification 70 Effects of GSH, GS-OCT, GS-HEX, bilirubin 70 and CDNB on DiAC modification of GST n The modification of GST n by EDAC 70 Kinetics of EDAC modification 70 Effects of GSH, GS-OCT, GS-HEX, bilirubin 71 and CDNB on EDAC inactivation of GST n Modification of GST n by DEPC 71 Detection of the reaction of GST n with DEPC 71 Determination of the numbers of histidine and 71 tyrosine residues of GST n modified by DEPC Modification by DEPC and enzyme activity 72 Effects of GSH or CDNB on DEPC modification of GST n 72 Peptide analysis of DEPC modification of GST n 72 N-terminal sequencing of GST n and 73 DEPC modified GST n Modification of GST n by NBS 73 74 Chemicals 74 Basic chemicals 74 Bacterial and cell culture reagents and media 74 Enzymes 74 Enzyme substrates and inhibitors 75 Hormones and vitamins 75 Oligonucleotides and nucleic acids 75 Radiochemicals 75 Reagent Kits 76 Other materials 76 6 Commonly used solutions 76 Bacterial cell culture 76 Bacterial strains 76 Bacterial growth media 77 Plasmids and cosmids 77 Isolation of plasmid DNA 77 Mini-scale isolation of plasmid DNA 77 Large scale isolation of plasmid DNA 78 Enzymatic modification of DNA 79 Restriction digestion 79 Exonuclease digestion 80 Treatment of DNA fragment with alkaline 80 phosphatase Isolation of restriction fragments 80 Agarose gel electrophoresis of DNA fragments 80 Isolation of restriction fragments from 81 agarose gels Southern blot analysis 81 Transfer of DNA fragments to solid support 81 Oligo-labelling of DNA fragment by 82 hexadeoxynucleotide primers Hybridization 82 Polymerase chain reaction (PCR) 83 Plasmid cloning of DNA fragments 83 Ligation 83 Preparation of competent cells 84 Transformation of E.coli JM83 84 Colony hybridization 84 DNA sequencing 85 Annealing template and primer 85 Labelling reactions 86 Termination reactions 86 Denaturing gel electrophoresis 86 Eukaryotic cell culture 87 Eukaryotic cell lines 87 7 Content 2.2.12.2 Routine cell culture 88 2.2.13 Transfection of plasmid DNA into eukaryotic 88 cell lines 2.2.13.1 Transfection 88 2.2.13.2 Chloramphenicol acetyl transferase (CAT) assay 89 2.2.13.3 Assay of 3-galactosidase activity 90 2.2.13.4 Determination of protein concentration 90 2.2.14 Slot blotting 91 2.2.14.1 RNA preparation from cultured cells 91 2.2.14.2 Blotting 91 2.2.14.3 RNA blot hybridization 92 2.2.15 Gel retardation 92 2.2.15.1 Preparation of cell nuclear extract 92 2.2.15.2 preparation of gel 93 2.2.15.3 End labelling of oligonucleotides 93 2.2.15.4 Formation of the DNA-protein complex 93 2.2.15.5 Electrophoresis 94 2.2.15.6 Autoradiography 94 2.2.16 Quantification of GST n in cultured cells 94 2.2.16.1 Preparation of cytosol from cultured cell 94 2.2.16.2 Analysis of GSTk subunit by reverse phase HPLC 95 CHAPTER III THE G-SITE OF GLUTATHIONE TRANSFERASE n 96 Forward 97 3.1 Cysteine residues 97 3.1.1 Modification of GST n by DTNB
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