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Glutathione S-Transferases and Oxidative Stress in Saccharomyces Cerevisiae

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Glutathione S-Transferases and Oxidative Stress in Saccharomyces Cerevisiae Thèse présentée pour obtenir le grade de Docteur de l’Université Louis Pasteur, Strasbourg I et de l’Université de Sofia St. Kliment Ohridski Discipline : Sciences du vivant Aspects moléculaires et cellulaires de la biologie Présentée par : Tatina Todorova TODOROVA Glutathione S-transferases and oxidative stress in Saccharomyces cerevisiae Soutenue publiquement le 20 juin 2007 Membres du jury: Directeur de thèse M. Stéphane VUILLEUMIER Professeur à l’Université Louis Pasteur, Strasbourg Directrice de thèse Mme Anna KUJUMDZIEVA Professeur Associé à l’Université de Sofia Président du jury Mme Marie-Claire LETT Professeur à l’Université Louis Pasteur, Strasbourg Rapporteur interne M. Ermanno CANDOLFI Professeur à l’Université Louis Pasteur, Strasbourg Rapporteur externe Mme Veneta GROUDEVA Professeur Associé à l’Université de Sofia Rapporteur externe M. Michel AIGLE Professeur à l’Université Claude Bernard, Lyon Acknowledgements This thesis was a cotutelle project between the University Louis Pasteur, Strasbourg and the Sofia University St. Kliment Ohridski. It would not have been possible without the financial support of the Agence Universitaire de la Francophonie, the European Doctoral College of Strasbourg and National Science Fund of Bulgarian Ministry of Education and Science (Project № Б-ВУ- 201/06). I am grateful to my supervisor Professor Stéphane Vuilleumier from the University Louis Pasteur for the constructive atmosphere during the work and for guiding my research by helpful scientific discussions. My sincere gratitude is due to Associate Professor Anna Kujumdzieva, my supervisor at the Sofia University. I wish to thank her for the support and advices. I want to acknowledge the reviewers of my thesis, Associate Professor Veneta Groudeva (Sofia University St. Kliment Ohridski), Professor Michel Aigle (University Claude Bernard, Lyon), Professor Ermanno Candolfi (University Louis Pasteur, Strasbourg) and Professor Marie-Claire Lett (University Louis Pasteur, Strasbourg) for critical reading of the thesis, and for their valuable comments and suggestions. I would like to thank to all my colleagues and friends in both laboratories for their support and help not only in the scientific matter but also in the everyday life. Finally, my warm thanks to my family and my friends for supporting me in so many ways. Ms Tatina Todorova was a member of the European Doctoral College of the Universities of Strasbourg during the preparation of her PhD thesis, from 2004 to 2007, class name René Cassin. She has benefited from specific financial supports offered by the College and, along with her mainstream research, has followed a special course on topics of general European interests presented by international experts. This PhD research project has been led with the collaboration of two universities: the University “St. Kliment Ohridski, Sofia, Bulgaria and the University Louis Pasteur, Strasbourg, France. Abbreviations used GSH – glutathione GSTs – glutathione S-transferases CDNB – 1-chloro-2,4-dinitrobenzene MAPEG – membrane associated proteins involved in eicosanoid and glutathione metabolism G-site – glutathione-binding site H-site – hydrophobic substrate binding site eEF – eukaryotic elongation factor Grx – glutaredoxin ROS – reactive oxygen species SOD – superoxide dismutase γ-GC – γ -glutamyl cysteine Gsh2 – glutathione synthetase Glr – glutathione reductase Trx – thioredoxin GPx – glutathione peroxidase PHGPx – phospholipid hydroperoxide glutathione peroxidase PUFA – polyunsaturated fatty acid 4-HNE – 4-hydroxynonenal MMA(V) – monomethylarsonic acid DMA(V) – dimethylarsinic acid MMA(III) – monomethylarsonous acid DMA(III) – dimethylarsinous acid MIP – major intrinsic protein NCR – nitrogen catabolite repression Acr2p – arsenate reductase Ycf1 – yeast cadmium factor MAPKs – mitogen-activated protein kinases JNK – c-Jun NH2-terminal kinase PKC – protein kinase C MAPKKs – mitogen-activated protein kinase kinases MAPKKKs – mitogen-activated protein kinase kinase kinases bZIP – basic region leucine zipper TOR – target of rapamycin Gdh1 – NADPH-dependent glutamate dehydrogenase Gdh2 – NAD+-dependent glutamate dehydrogenase Gln1 – glutamine synthetase CONTENTS INTRODUCTION 4 I. The glutathione S-transferase family of enzymes: essential catalysts of cellular detoxification 5 1. General characteristics and functions of glutathione S-transferases 6 1.1. Overview of cell detoxification 6 1.2. Detoxification function of glutathione S-transferases 6 1.3. Classification of enzymes within the glutathione S-transferase family 8 1.4. Structural characteristics of GST enzymes 11 1.4.1. Cytosolic GSTs 11 1.4.2. Microsomal GSTs 14 1.4.3. Plasmid-encoded bacterial fosfomycin-resistance GSTs 14 1.4.4. Kappa class GSTs 15 1.5. Regulation of GST enzymes 15 1.6. Evolution of the GST enzyme family 16 2. Glutathione S-transferase-like proteins in yeast 16 2.1. The diversity of microbial GSTs 16 2.2. GSTs of fungi and yeasts 17 2.3. GSTs of known function in Saccharomyces cerevisiae 18 2.3.1. Gtt1 and Gtt2 18 2.3.2. Tef3p and Tef4p 19 2.3.3. Ure2p 20 2.3.4. Omega class GSTs 21 II. Antioxidant role of glutathione S-transferases 23 1. Oxidative stress defense in yeast 24 1.1. Oxygen metabolism in aerobic organisms 24 1.2. Main antioxidant defense systems in yeast 25 1.2.1. Non-enzymatic defense system 26 1.2.2. Enzymatic antioxidant defense system 28 2. The role of GSTs in the response against oxidative stress 31 2.1. The glutathione peroxidase function of GSTs 32 2.1.1. Protection role of GSTs against lipid peroxidation in mammals 32 2.1.2. Role of yeast GSTs in oxidative stress response 34 1 2.2. Role of GSTs in oxidative stress caused by arsenic species 34 2.2.1. Modes of arsenic action 34 2.2.2. Arsenic detoxification in mammals: specific role of GSTs 36 2.2.3. Mechanism of arsenic detoxification in yeast 37 III. Regulatory roles of GSTs: entering new territory 41 1. Introduction 42 2. Indirect regulatory role of GSTs 43 3. GSTs as direct regulators of cell signalling 45 3.1. GSTs modulate JNK signaling cascade 45 3.2. Ure2p: an essential regulator of GATA signaling pathway in yeast 49 3.2.1. GATA factor family 49 3.2.2. The GST Ure2p inhibits gene expression by GATA factors 50 3.2.3. Mechanism of GATA signal transduction 51 3.2.4. GATA regulation beyond nitrogen catabolite repression 53 3.3. GST regulation in mammals and yeast: common threads? 54 OBJECTIVES AND OUTLINE OF THE THESIS 57 RESULTS 60 I. Screening of GST mutant bank 61 Résumé en français 62 1. Introduction 64 2. Results 64 2.1. Identification of S. cerevisiae GST mutants sensitive to arsenic and to hydrogen peroxide 64 2.2. The ycf1Δ mutant is sensitive to As(V) and As(III) 72 2.3. The gpx3Δ mutant is sensitive to H2O2 73 2.4. The glr1Δ mutant is sensitive to H2O2 and As(V) 74 2.5. The grx5Δ mutant is hypersensitive to H2O2, As(V) and As(III) 76 3. Conclusion and perspectives 77 Annex 1. Materials and methods 78 II. Characterisation of the arsenic and peroxide sensitivity of the tef4Δ mutant of S. cerevisiae 79 Résumé en français 80 1. Introduction 81 2. Results 84 2 2.1. Phenotypic characterization of the tef4Δ mutant 84 2.2. Cloning and expression of TEF4 gene 88 Annex 2. Materials and methods 92 III. Regulation of arsenite uptake – a novel non-enzymatic role for the glutathione S-transferase Ure2 of Saccharomyces cerevisiae 97 Résumé en français 98 1. Introduction 100 2. Materials and methods 102 3. Results 105 3.1. The GST domain of Ure2p confers arsenite resistance in Saccharomyces cerevisiae 105 3.2. Effect of arsenic exposure on glutathione peroxidase activity, NCR-regulated enzyme activities and glutathione content in ure2Δ mutant 107 3.3. GATA regulation of arsenic sensitivity in S. cerevisiae 109 4. Discussion 115 IV. Oxidant response in ure2Δ mutant of Saccharomyces cerevisiae 121 Résumé en français 122 1. Introduction 124 2. Materials and methods 125 3. Results and discussion 128 3.1. Sensitivity of URE2 disruption mutant to oxidants 128 3.2. Effect of glutathione on the sensitivity of mutant ure2Δ to H2O2 131 3.3. Glutathione redox ratio and glutathione peroxidase activity in ure2Δ mutant 132 3.4. Catalase and superoxide dismutase activities in the ure2Δ mutant 134 3.5. Accumulation of reactive oxygen species in ure2Δ mutant 136 GENERAL SUMMARY AND PERSPECTIVES 138 REFERENCES 142 3 Introduction 4 Chapter 1 The glutathione S-transferase family of enzymes: essential catalysts of cellular detoxification 5 1. General characteristics and functions of glutathione S-transferases 1.1. Overview of cell detoxification Living organisms are continuously exposed to exogenous and endogenous toxic chemical species, which may cause adverse and sometimes lethal effects. The ability of living organisms to survive the risk posed by such compounds represents a fundamental biological adaptation for survival. Different strategies have been adopted by cells to counter the effect of toxic compounds and their metabolites. Defense mechanisms are usually general, rather than specific for a given chemical or organism. Among the defense mechanisms, such as sequestration and binding, catalytic biotransformation was evolved as a crucial mode of protection against toxic chemical species (Sheehan et al., 2001). Cells possess a broad ensemble of enzymes capable of transforming a wide range of different chemical structures and functionalities. The enzymatic detoxification of xenobiotics has been classified into three distinct phases, which act in a tightly integrated manner. Phases I and II enzymes catalyze the conversion of a lipophilic, non-polar xenobiotic into a more water-soluble and therefore less toxic metabolite, which can then be eliminated more easily from the cell. Phase I of detoxification is mainly the result of action by the cytochrome P450 system and mixed function oxidases (Ishikawa et al., 1997). These proteins are responsible for a wide range of reactions, of which oxidations appear to be the most important (Guengerich, 2001). Phase II enzymes catalyze the conjugation of activated xenobiotics to an endogenous water- soluble substrate, such as reduced glutathione (GSH) or uridine diphosphate (UDP)-glucuronic acid.
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