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Evolution and cellular resistance mechanisms of the Immunity-Related GTPases [Elektronische Ressource] / vorgelegt von Julia Hunn

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207 pages
Evolution and Cellular Resistance Mechanisms of the Immunity-Related GTPases Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Julia Hunn aus Singen (Htwl.) Köln 2007 Berichterstatter: Prof. Dr. Jonathan C. Howard Prof. Dr. Thomas Langer Prof. Dr. Otto Haller Tag der mündlichen Prüfung: 7. February 2008 Success is nothing more than going from failure to failure with undiminished optimism. Winston Churchill TABLE OF CONTENTS Table of contents I. INTRODUCTION 1 I.1. INFECTION, IMMUNITY AND CYTOKINES 1 I.2. INTERFERONS 1 I.3. IFN-MEDIATED CELL AUTONOMOUS RESISTANCE 4 I.4. GUANOSINE TRIPHOSPHATASES (GTPASES) 4 I.4.1. Dynamin 6 I.4.2. Large, interferon-inducible GTPases 7 I.5. TOXOPLASMA GONDII 19 I.6. THE AIM OF THIS STUDY 22 II. MATERIAL AND METHODS 24 II.1. MATERIAL 24 II.1.1. Mammalian cells and media 24 II.1.2. Bacterial strains and media 24 II.1.3. Yeast strains and media 24 II.1.4. Toxoplasma gondii strains 25 II.1.5. Vectors 25 II.1.6. Generation of expression constructs 25 II.1.7. Primers 27 II.1.8. Primary immunoreagents 27 II.1.9. Secondary immunoreagents 28 II.2.
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Evolution and Cellular Resistance Mechanisms
of the Immunity-Related GTPases















Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln





vorgelegt von
Julia Hunn
aus Singen (Htwl.)

Köln 2007































Berichterstatter: Prof. Dr. Jonathan C. Howard
Prof. Dr. Thomas Langer
Prof. Dr. Otto Haller

Tag der mündlichen Prüfung: 7. February 2008





































Success is nothing more than going from failure to failure with undiminished optimism.
Winston Churchill
TABLE OF CONTENTS
Table of contents
I. INTRODUCTION 1
I.1. INFECTION, IMMUNITY AND CYTOKINES 1
I.2. INTERFERONS 1
I.3. IFN-MEDIATED CELL AUTONOMOUS RESISTANCE 4
I.4. GUANOSINE TRIPHOSPHATASES (GTPASES) 4
I.4.1. Dynamin 6
I.4.2. Large, interferon-inducible GTPases 7
I.5. TOXOPLASMA GONDII 19
I.6. THE AIM OF THIS STUDY 22
II. MATERIAL AND METHODS 24
II.1. MATERIAL 24
II.1.1. Mammalian cells and media 24
II.1.2. Bacterial strains and media 24
II.1.3. Yeast strains and media 24
II.1.4. Toxoplasma gondii strains 25
II.1.5. Vectors 25
II.1.6. Generation of expression constructs 25
II.1.7. Primers 27
II.1.8. Primary immunoreagents 27
II.1.9. Secondary immunoreagents 28
II.2. METHODS: PHYLOGENETIC ANALYSIS 29
II.2.1. Use of database resources 29
II.2.2. Generation of multisequence alignment and phylogenetic trees 29
II.3. METHODS: MUTAGENESIS AND CLONING 29
II.3.1. Preparation of chemical competent bacteria 29
II.3.2. Transformation of competent bacteria 29
II.3.3. Plasmid DNA isolation 30
II.3.4. Agarose gel electrophoresis 30
II.3.5. Purification of DNA fragments from agarose gel 30
II.3.6. Restriction digest 30
II.3.7. Ligation 30
II.3.8. Sequencing 30
II.3.9. Site directed mutagenesis 31
II.4. METHODS: PROTEIN BIOCHEMISTRY 31
II.4.1. Expression and purification of recombinant protein 31
II.4.2. Guanine nucleotide binding parameters 32
II.4.3. GTP hydrolysis assay 32
II.4.4. Analysis of protein oligomerisation by light scattering 32
II.5. METHODS: MAMMALIAN CELLS 33
II.5.1. Freezing and thawing of mammalian cells 33
II.5.2. Transfection of mammalian cells 33
II.5.3. Hormone-inducible mammalian expression system (GeneSwitch) 33
II.5.4. Generation of stable inducible cell lines 34
II.5.5. Induction with IFNγ and Mifepristone 34
II.5.6. Immunofluorescence 35
II.5.7. Cell proliferation assay 35
II.5.8. Cell cycle assay 35
II.6. METHODS: ANALYSIS OF CELLULAR PROTEIN 35
II.6.1. Generation of cell lysates for SDS-PAGE 35
II.6.2. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) 36
II.6.3. Western Blotting and Ponceau S staining 36
II.6.4. Sequential Triton X-114 partitioning assay 36
II.6.5. Co-Immunoprecipitation 37
II.6.6. Pull down 37
II.6.7. Coomassie staining 38
II.6.8. Analytical size exclusion chromatograpgy of cellular IRG GTPases 38
II.7. METHODS: INFECTION ASSAYS 38
II.7.1. In vitro passage of Toxoplasma gondii 38
II.7.2. Infection of murine cells with Toxoplasma gondii 39
II.7.3. Quantication of IRG signals on T. gondii parasitophorous vacuoles 39 TABLE OF CONTENTS
II.8. METHODS: YEAST 2 HYBRID 39
II.8.1. Lithium acetate transformation of Saccharomyces cerevisiae 39
II.8.2. Analysis of protein interaction by yeast two-hybrid (Y2H) 39
III. RESULTS 41
III.1. GENOMIC ORGANISATION, SYNTENY AND PHYLOGENETIC RELATIONSHIP
OF IRG GTPASES 41
III.1.1. Nomenclature 41
III.1.2. IRG genes of the C57BL/6 mouse 43
III.1.3. Human IRG genes and their synteny relationship to mouse IRGs 52
III.1.4. IRG homologues in rodents and lagomorphs (Glires) 59
III.1.5. IRG homologues in the carnivore Canis familiaris 60
III.1.6. IRG homologues in other mammalian species 67
III.1.7. IRG homoin non-human primates 69
III.1.8. IRG homologues outside the Eutheria 71
III.2. REGULATORY INTERACTIONS BETWEEN IRG GTPASES CONTROLLING
ACTIVATION AND FUNCTION 80
III.2.1. Generation and characterisation of stable cell lines inducibly expressing
single IRGs 80
III.2.2. Influence of IRG expression cell proliferation and survival 84
III.2.3. Influence of IFNγ on the subcellular localisation of Irgm1-3, Irgc, Irgd
and Irga6 86
III.2.4. Biochemical properties of Irga6 G1 mutants 94
III.2.5. Influence of nucleotide binding on the subcellular localisation of Irga6 96
III.2.6. e IFNγ and nucleotide on the subcellular localisation of Irgb6 97
III.2.7. Behaviour of IRG proteins in size exclusion chromatography 99
III.2.8. Regulation of Irga6 and Irgb6 positioning by the GMS proteins 101
III.2.9. Nucleotide-dependent direct interactions of IRG proteins in Y2H 104
III.2.10. Direct GDP-dependent interaction of cellular Irgm3 with Irga6 106
III.2.11. Regulation of Irga6 in Toxoplasma-infected cells 109
III.2.12. Regulation of Irgb6, Irgd, Irgm1-3 and Irgc in Toxoplasma-infected cells113
III.2.13. Virulent T. gondii counteract IRG protein accumulation at the PVM 116
IV. DISCUSSION 118
IV.1. SIGNS FOR DIVERGENT EVOLUTION OF THE IRG GENES IN RODENTS 118
IV.2. DIVERGENT IRG GENES ARE FOUND THROUGHOUT THE MAMMALS 119
IV.3. LOSS OF IRG RESISTANCE SYSTEM IN THE ANTHROPOIDS 121
IV.4. THE IRG FAMILY IS ANCIENT AND PRESENT IN EUCHORDATES 123
IV.5. IRG PROTEINS IN CELL PROLIFERATION AND SURVIVAL 126
IV.6. COVALENT PROTEIN MODIFICATION OF IRGA6 126
IV.7. IRG LOCALISATION IS REGULATED BY IFN AND NUCLEOTIDE 128
IV.8. IRGA6 AND IRGB6 AGGREGATES FORMED IN ABSENCE OF IFNγ
REPRESENT GTP-BOUND HOMOMERS 130
IV.9. GTP-BOUND HOMOMERIC IRGA6 AND IRGB6 COMPLEXES
DISSOCIATE EX VIVO 131
IV.10. THE THREE GMS PROTEINS NEGATIVELY REGULATE IRGA6 AND IRGB6
131
IV.11. DIRECT, NUCLEOTIDE-DEPENDENT INTERACTIONS OF IRG ROTEINS 132
IV.12. DIRECT, GDP-DEPENDENT INTERACTION OF IRGA6 WITH IRGM3
OCCURS VIA THE G-DOMAIN 134
IV.13. REGULATORY IRG INTERACTIONS IN TOXOPLASMA INFECTION 135
IV.14. IRGC – A PROTEIN IN SEARCH OF A FUNCTION 138
IV.15. VIRULENT TOXOPLASMA GONDII INHIBIT IRG PROTEINS 139
IV.16. MODEL OF IRG FUNCTION IN UNINFECTED AND INFECTED CELLS 140
V. APPENDIX 142
V.1. PURIFICATION OF RECOMBINANT IRGA6(S83N) AND (K82A) ROTEIN 142
V.2. EXPRESSION VECTORS 143
V.3. DNA SEQUENCE OF THE EXPRESSED MURINE IRGS 144
V.4. ABBREVIATION OF SPECIES NAMES 145
V.5. SEQUENCE SOURCES OF MOUSE AND HUMAN IRG GENES 146
V.6. NUCLEOTIDE ALIGNMENT OF MOUSE IRGA6 AND IRGA6* C57BL/6) 148 TABLE OF CONTENTS
V.7. PROTEIN AND NUCLEOTIDE ALIGNMENT OF MOUSE IRGB3 AND IRGB4 149
V.8. ALIGNMENT OF MAMMALIAN IRGC PROTEINS 150
V.9. ALIGN N IRGQ 151
V.10. ALIGNMENT OF ORANG-UTAN IRGM SEQUENCES (PONGO PYGMAEUS) 152
V.11. ALIG TAKIFUGU RUBRIPES IRG PROTEINS 152
V.12. ALIG T 153
V.13. IRG PROTEIN SEQUENCES 153
VI. REFERENCES 174
VII. SUMMARY 192
VIII. ZUSAMMENFASSUNG 193
IX. ACKNOWLEDGEMENTS 194
X. KOLLABORATIONEN 195
XI. ERKLÄRUNG 196
XII. LEBENSLAUF 197
ABBREVIATIONS
Abbreviations
AD activation domain GPI glycosyl-phosphatidylinositol
ADAR adenosine deaminase acting on RNA GppNHp 5’-guanylylimidodiphosphate
ADRP adipocyte differentiation-related GRA dense granule protein
protein GST glutathione-S-transferase
APOBEC3G apolipoprotein B mRNA-editing GTP guanosine triphosphate
enzyme, catalytic polypeptide-like 3G GTPaseguanosinetriphosphatase
APS ammonium peroxide sulfate GTPγS guanosine 5'-(3-O-thio)triphosphate
ATP adenosine triphosphate GTPI GTPase IFN-induced
ATPase adenosine triphosphatase HRP horse radisch peroxidase
BAC bacterial artificial chromosome HSV-1 herpes simplex virus type 1
BD DNA binding domain IBDV infectious bursal disease virus
BLAST basic local alignment search tool IDO indoleamine 2,3-dioxygenase
BSA bovine serum albumin IF immunofluorescence
CARD caspase recruitment domain IFN interferon
CID central interacting domain IFNAR IFN α receptor
CIITA MHC class II transactivator (CIITA) IFNGR IFN γ
DAPI 4',6-Diamidine-2'-phenylindole IFNLR IFN λ receptor
dihydrochloride IGTP inducibly expressed GTPase
DC dentritic cell IIGP IFN-inducible GTPase
DBD DNA binding domain IL interleukin
DMEM Dulbecco’s modified Eagle’s medium IL10R2 interleukin 10 receptor 2
DMSO dimethylsulfoxid iNOS inducible nitric oxide synthase
Ds double stranded IPTG isopropyl-β-D thiogalactoside
DTT dithiothreitol IRF IFN regulatory factor
ECMV encephalomyocarditis virus IRG immunity-related GTPases
EDTA ethylen diamine tetraacetate IRG-47 IFN-regulated GTPase 47 kDa
EF-G elongation factor G ISGF3 IFN-stimulated gene factor 3
EF-Tu elongation factor thermal unstable ISG15 ulated gene 15
EGFP enhanced green fluorescent protein ISG20 IFN-stimulated 3’-5’ exonuclease
ER endoplasmic reticulum gene 20
eRF1 elongation release factor 1 ISRE IFN-stimulated response element
ERV9 endogenous retrovirus 9 Jak Janus kinase
EST expressed sequence tag LD lethal dose
EtOH ethanol LPS lipopolysaccharide
FAT10 HLA-F adjacent transcript 10 LRG-47 LPS-regulated GTPase 47 kDa
FCS fetal calf serum LTR long terminal repeat
FLUAV influenza A virus LZ leucine zipper
FTase farnesyl transferase MEC mouse oviduct epithelial cell
FPLC fast protein liquid chromatography MEF mouse embryonic fibroblast
G domain GTPase domain mGDP mant guanosine diphosphat
GAF gamma activated factor mGTPγS mant GTPγS
GAL4 galactose induced gene 4 MHC major histocompatibility complex
GAS gamma-activated sequence MIC microneme protein
GAP GTPase activating protein Mif Mifepristone
GBP guanylate binding protein MOI multiplicity of infection
GDI guanine nucleotide dissociation MW molecular weight
inhibitor
GDP guanosine diphosphate MxA Myxovirus resistance protein A
GDP βS guanosine-[ β-thio]-diphosphate NEDD8 neural precursor cell expressed,
GED GTPase effector domain developmentally down-regulated 8
GEF guanin nucleotide exchange factor NK cell natural killer cell
GFP green fluorescent protein NMT N-myristoyl transferase
GGTase geranylgeranyl transferase NOS2 nitric oxide synthase 2
ABBREVIATIONS

NRAMP1 natural resistance-associated Sox Sry-type high-mobility-group
macrophage protein 1 domain box
2’-5’ OAS 2’-5’-oligo-adenylate synthetase SRP signal recognition particle
o.n. over night SSAHA sequence search and alignment by
OD optical density Hashing algorithm
ORF open reading frame STAT signal transducer and activator of
PAGE polyacrylamind gel electrophoresis transcription
PBS phosphate buffered saline SUMO small ubiquitin-related modifier
PH pleckstrin homology TGTP T cell specific GTPase
phox phagocyte oxidase TIM triose-phosphate isomerase
PKR dsRNA-dependent protein kinase TK HSV thymidine kinase
PML promyelocytic leukemia TNF α tumor necrosis factor α
PMSF phenylmethylsulphonyl fluoride TRIM5α tripartite motif protein 5α
PRD proline-rich domain tsvGORASP testis specific splice variant Golgi
PV parasitophorous vacuole reassembly and stacking protein 2
PVM parasitophorous vacuolar membrane Tyk2 tyrosine kinase 2
Rab Ras genes from rat brain U units
Ran Ras related nuclear protein UAS upstream activating sequence
Ras rat sarcoma URG4 up-regulated gene 5
Rho a homologue UTR untranslated region
RNAi RNA interference VLIG very large inducible GTPase
RON rhoptry neck protein VSV vesicular stomatitis virus
ROP protein WB Western blot
ROS reactive oxygen sspecies Y2H yeast two-hybrid
RT room temperature YFP yellow fluorescent protein
SD synthetic defined YPD yeast-peptone-dextrose medium
SDS sodium dodecylsulfate ZAP zinc-finger antiviral protein