Thèse présentée pour obtenir le grade de Docteur

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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

  • stress response

  • phospholipid hydroperoxi de glutathione peroxidase

  • kinase kinase

  • gsts

  • glutathione

  • mitogen activated protein

  • oxidative stress

  • gsts against


Source : scd-theses.u-strasbg.fr
Nombre de pages : 160
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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 Louig
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 NH -terminal kinase 2PKC – 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 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 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
12.2. 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 ycf1 Δ mutant is sensitive to As(V) and As(III) 72
2.3. The gpx3Δ mutant is sensitive to H O 73 22
2.4. The glr1 Δ mutant is sensitive to H O and As(V) 74 2 2
2.5. The grx5 Δ mutant is hypersensitive to H O , As(V) and As(III) 76 2 2
3. Conclusion and perspectives 77
Annex 1. Materials and methods 78
II. Characterisation of the arsenic and peroxide sensitivity of the tef4 Δ
mutant of S. cerevisiae 79
Résumé en français 80
1. Introduction 81
2. Results 84
22.1. Phenotypic characterization of the tef4 Δ 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 ure2 Δ mutant 107
3.3. GATA regulation of arsenic sensitivity in S. cerevisiae 109
4. Discussion 115
IV. Oxidant response in ure2 Δ 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 ure2 Δ to H O 131 22
3.3. Glutathione redox ratio and glutathione peroxidase activity in ure2 Δ mutant 132
3.4. Catalase and superoxide dismutase activities in the ure2 Δ mutant 134
3.5. Accumulation of reactive oxygen species in ure2 Δ 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














51. 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. Quantitatively, conjugation to GSH, which is catalyzed by glutathione S-transferases
(GSTs) is the major phase II reaction in many species (Sheehan et al., 2001).
The resulting more soluble compounds are eliminated from cells in phase III of the
detoxification process. In eukaryotic organisms, they are actively excreted or compartmentalized
in the vacuole by ATP-dependent GS-X pumps (Ishikawa et al., 1997; Ernst et al., 2005).
Indeed, as the glutathionylated moiety is hydrophilic, the conjugate cannot usually simply re-
diffuse back into the cell.

1.2. Detoxification function of glutathione S-transferases
Glutathione S-transferases promote the inactivation and degradation of a wide range of
compounds by the formation of glutathione conjugates. Enzymes of this family are generally
characterized by broad substrate specificity and low affinity (high Km values). A low catalytic
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