Addressing the role of mitochondrial thioredoxin reductase and xCT in the maintenance of redox homeostasis [Elektronische Ressource] / Tamara Perisic

Tamara Perisic

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 59
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

A thesis submitted for the Doctoral degree (Dr. rer. nat.) to the Faculty of Biology at the
Luidwig-Maximilians-University, Munich, Germany







Addressing the Role of Mitochondrial Thioredoxin Reductase
and xCT in the Maintenance of Redox Homeostasis




Tamara Perisic





Helmholtz Zentrum München, German Reasearch Center for Environmental Health
Institute of Clinical Molecular Biology and Tumor Genetics



Munich, October 2009
Ehrenwörtliche Versicherung

Ich versichere hiermit ehrenwörtlich, dass die Dissertation von mir selbständig,
ohne unerlaubte Beihilfe angefertigt ist.
Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen
Prüfungskommission eingereicht.
Ich habe noch zu keinem früheren Zeitpunkt versucht eine Dissertation
einzureichen oder an einer Doktorprüfung teilzunehmen.
.....................................................................
(Tamara Perisic)



Dissertation eingereicht am 8. Oktober 2009

1. Gutachter: Prof. Dr. Dirk Eick
2. Gutachter: Dr. Daniel Krappmann
3. Gutachter: Prof. Dr. Hans-Ulrich Koop
4. Protokoll: Prof. Dr. Michael Boshart






Tag der mündlichen Prüfung: 23. 03. 2010



Table of contents

LIST OF ABREVIATIONS I
1. INTRODUCTION 1
1.1. Biological sources of ROS and its role in diverse biological processes 1
1.2. Selenoproteins 2
1.2.1. Incorporation of selenium 3
1.2.2. Family of thioredoxin reductases 5
1.2.3 Mammalian thioredoxin system 7
1.3. Peroxiredoxins 13
1.3.1. The catalytic mechanisms of peroxiredoxins 15
1.3.2. The role of sulfiredoxin (Srx) 17
−1.4. System x 19 C
1.5. Objectives 21
2. MATERIALS AND METHODS 23
2.1. Materials
RT-PCR 28
Expression and Targeting Vectors
2.2. Methods 31
2.2.1. Cell culture 31
2.2.2.Gene-transfer methods 34
2.2.3. Molecular biology techniques 35
2.2.4. Flow cytometry 46
2.2.5. Biochemical methods 49
2.2.6. ES cell technology 50
I 3. RESULTS 53
3.1. Functional analysis of mitochondrial thioredoxin reductase (Txnrd2) 53
3.1.1. Phenotype of primary MEFs lacking Txnrd2 53
3.1.3. Antioxidants rescue loss of Txnrd2 54
3.1.4. Intracellular ROS are augmented in cells lacking Txnrd2 55
−/ −3.1.5. Susceptibility of Txnrd2 cells towards pro-oxidants and genotoxic agents 56
−/ −3.1.6. Sensitivity of Txnrd2 cells towards inhibition of de novo GSH biosynthesis 59
−/ −3.1.7. Expression of mitochondrial peroxiredoxins is increased in Txnrd2 cells 61
3.1.8. Time-dependent increase of Prx III levels in response to H O 62 2 2
−/ −3.1.9. Ectopic expression of Txnrd2 and Txnrd1 in Txnrd2 cells 64
3.1.10. Stable expression of Txnrd1 in Txnrd2 knock-out cells 65
−/ −3.1.11. Add-back of mitoTxnrd2 rescues Txnrd2 cells from cell death induced by
GSH depletion 67
3.1.12. The role of Txnrd2 in normal physiology and disease 69
−3.2. The role of system x in maintenance of intracellular GSH levels and C
cystine/cysteine redox balance in cultured cells 75
−3.2.1. The protective role of system x in Burkitt lymphoma (BL) cells 75 C
3.2.2. The thioredoxin system as the possible driving force for the cystine/cysteine
redox cycle 76
3.3. Generation of conditional xCT-knock-in mice 80
3.3.1 Gene targeting in ES cells 80
3.3.2. Analysis of the functionality of the neoR-STOP-xCT cassette 82
flSTOP/CreERT23.3.3. Generation of R26mxCT mice 82
3.3.4. Breeding of xCT-transgenic mice with the Tam-inducible CreERT2-Deleter
mouse 84
flSTOP/CreERT23.3.4. Phenotypic analysis of R26mxCT mice 84
4. DISCUSSION 93
4.1. Absence of Txnrd2 causes high ROS accumulation, increased sensitivity
towards pro-oxidants and impaired cell growth 93
II 4.2. Compensatory up-regulation of mitochondrial peroxiredoxins III and V in
response to Txnrd2 disruption 98
4.3. Txnrd1 and Txnrd2 do not show functional redundancy in vivo 99
4.4. Txnrd2 is dispensable for maintaining the Cys/(Cys) cycle 101 2
4.5. Txnrd2 protects myocardial tissue from oxidative damage and is implicated in
the pathogenesis of Dilatative Cardiomyopathy 103
4.6. Inducible overexpression of xCT in mice is not protective, but causes spleen,
thymus and testis atrophy and defective erythropoiesis 104
5. SUMMARY 109
6. REFERENCES 111
7. APPENDIX 129
7.1. Acknowledgements
7.2. Curriculum vitae 131

III List of Abreviations

2-ME β-mercaptoethanol
Amp Ampicillin
AmpR resistance gene (ß-lactamase)
AMV Avian myeloblastosis virus
ATP Adenosine-5'-triphosphate
BOOH t -butylhydroperoxide
BSA Bovine serum albumin
BSO L-buthionine sulfoximine
CDK Cycline dependent kinase
CMV Cytomegalovirus
Cre Cre recombinase
Cys Cysteine
(Cys) Cystine (oxidised Cys dimer) 2
DAPI 4´,6-diamidino-2-phenylindole, dihydrochloride
DCFH-DA Dichlorofluorescin diacetate
DCM Dilated cardiomyopathy
DIOs Iodothyronine deiodinases
DMEM Dulbecco's Modified Eagle Medium
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide
DTT Dithiothreitol
EDTA Ethylenediamine-N,N,N’,N’-tetra-acetic acid
EFSec Sec-specific elongation factor
EGF Epidermal growth factor
eGFP Enhanced green fluorescence protein
EpRE Electrophile response element
ERK Extracellular signal-regulated kinase
ES cells Embryonic stem cells
FACS Fluorescence activated cell sorting
i FAD Flavin adenine dinucleotide
FCS Fetal calf serum
GpAATs Glycoprotein-associated amino acid transporters
GPx Glutathione peroxidase
GR reductase
GRx Glutaredoxin
GSH
GSS synthetase
GSSG Oxidized glutathione
GTP Guanosine-5'-triphosphate
H O Hydrogen peroxide 2 2
HO• Hydroxyl radical
HO• Hydroperoxy radical 2
HPRT Hypoxanthine-guanine phosphoribosyltransferase
IRES Internal ribosomal entry site
LMP agarose Low melting point agarose
LTR Long terminal repeat
MAPK mitogen activated protein kinase
MC MERCreMER
MEFs Murine embryonic fibroblasts
MitoPY Mitochondrial Peroxi-yellow
MLS leader sequence
NAC N-Acetyl-L-cysteine
NADPH Nicotinamide adenine dinucleotide phosphate
NeoR Neomycin phosphotransferase
NESs Nuclear export signals
NF κB factor κB
NLS localization signal
Nrf2 NF-E2-related factor 2
NTAPe-tag N-terminal tandem affinity purification enhanced tag
nucmemb Nuclear membrane anchor
2−O Superoxide anion
OVA Ovalbumin
pA PolyA signal
ii PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PDGF Platelet-derived growth factor
PEITC Phenyl ethyl isothiocyanate
PFA Paraformaldehyde
PI Propidium iodide
PRE Post regulatory element
Prx Peroxiredoxin
PS Phosphatidylserine
PuroR Puromycin N-acetyltransferase gene
Py ori Polyoma origin of replication
PYAM Peroxi-yellow acetoxy methyl-ester
PYME methyl-ester
RFP Red fluorescence protein
RNA Ribonucleic acid
RO• Alkoxyl radical
RO• Perl ril 2
ROS Reactive oxygen species
rpL30 Ribosomal protein L30
rT3 Reverse-triiodothyronine
SA Splice acceptor
SBP2 SECIS-binding protein 2
SDS Sodium dodecyl sulfate
Se Selenium
Sec Selenocysteine
SECIS insertion sequence
Secp43 43 kDa RNA-binding protein
-SeH Selenol group
SFFV Spleen focus forming virus
SF-tag Strep-FLAG-tag
SIN Self-inactivating 3’ LTR
SLA Soluble liver antigen protein
SOD Superoxide dismutase
Srx Sulfiredoxin
iii SV40 SV-40 promoter
T3 Triiodothyronine
T4 Thyroxine
Tam Tamoxifen
TGR Thioredoxin-glutathione reductase (Txnrd3)
TNF Tumour necrosis factor
TPx Thioredoxin peroxidases
Trsp Selenocysteine-specific tRNA gene
Trx
Txnrd Murine thioredoxin reductase
TXNRD Human
UGA “opal“ stop codon
wt Wild-type
γ-GCS γ-glutamylcysteine synthetase

iv Introduction
1. Introduction

1.1. Biological sources of ROS and its role in diverse biological
processes

2–Reactive oxygen species (ROS) is a collective term and includes superoxide anion (O ),
hydrogen peroxide (H O ), hydroxyl radical (HO•), hydroperoxy radical (HO •), peroxyl radical 2 2 2
(RO •) and alkoxyl radical (RO•). Among them, hydrogen peroxide (H O ) is the most 2 2 2
extensively studied one. Most biological sources of H O involve the spontaneous or catalytic 2 2
2−breakdown of superoxide anion (O ), generated by the partial reduction of oxygen during
mitochondrial respiration (Boveris, 1984) . Also, exposure of cells to a variety of physical,
chemical, and biological agents causes