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Identification and Characterization of HAX1 Interacting Proteins

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Identification and Characterization of HAX1 Interacting Proteins Identification and Characterization of HAX1 Interacting proteins Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von M.Sc. (Biosciences), Eswarappa Pradeep Bulagonda geboren am 14. April 1981 in Narpala/Ananthapur, Indien Hannover 2009 1 Referent: Prof. Dr. Matthias Gaestel Korreferent: Prof. Dr. Christoph Klein Tag der Promotion: 01.02.2010 2 An Offering At thy lotus feet 3 Zusammenfassung Das “Kostman Gen” kodiert ein Protein, das in der Literatur als HAX-1 bezeichnet wird und mit dem HS-1 Protein X, ein mitochondriales 35kDA grosses intrazelluläres Protein, assoziiert ist. Bif-1, Teil der SH3 Domänenfamilie der Endophilin und Serpin b1a Gruppe der Ova-Serpin Superfamilie wurde mittels eines Yeast-Two-Hybrid Screens, als mit HAX-1 interagierendes Protein identifiziert. Eine Überexpression von HAX-1 in E.coli war aufgrund der möglichen Präsenz von seltenen Codons und Codon Bias nicht möglich. Ferner war die Überexpression von HAX-1 in Dictyostelium discoideum aufgrund der gegebenen Instabilität dieses Proteins nicht zu erreichen. Es konnte gezeigt werden, dass Bif-1 und HAX-1 sowohl in 293T als auch in Jurkat Zellen endogen miteinander interagieren. Und dass der C-Terminus von HAX-1, Aminosäuren 179 bis 234, in diese Bindung mit Bif-1 involviert ist. Im Cytoplasma konnte eine signifikante Menge von endogenem HAX-1 und Bif-1 nachgewiesen werden, die dort Co-Lokalisiert vorlagen. Eine EBSS-Starvation führte in HAX-1 defizienten Patientenfibroblasten durch Autophagie zum Zelltod. Ferner belegen die Daten, dass eine EBSS-Starvation in HAX-1 defizienten Zellen zu einem Anstieg des Bif-1 Levels und dessen phosphorylierter Form führte. Zusammenfassend kann festgestellt werden, dass die vorliegende Studie erstmalig die gemeinsame Interaktion von Hax-1 und Bif-1 belegt und diese, die durch Autophagie hervorgerufenen Zelltod, nach EBSS-Starvation reguliert. HAX1: Hematopoietic Cell Specific Lineage substrate 1 associated protein X 1 Bif-1: BAX interacting Factor 1 EBSS: Earle’s Balanced Salt solution 4 Synopsis The ‘Kostmann gene’ encodes a protein designated as HAX-1, for HS-1-associated protein X, a mitochondria targeted protein (35KDa intracellular protein). Bif-1 member of the SH3 domain family of endophilins and Serpin b1a member of the Ova- Serpin superfamily were fished out by the Yeast Tow hybrid screen using HAX1 as bait. Over expression of HAX1 in E.coli was not possible probably because of the presence of rare codons and codon bias. HAX1 was found to be unstable when overexpressed and purified in Dictyostelium discoideum. Bif-1 and HAX1 were found to interact with each other endogenously in 293T and Jurkat cell lines The C-terminal 179 to 234 amino acids of HAX1 were proved to be involved in binding to Bif-1. Significant amounts of endogenous HAX1 and Bif-1 were found to Co- localize in the cytoplasm. Loss of HAX1 leads to autophagic cell death upon EBSS starvation in HAX1 deficient patient fibroblasts. Loss of HAX1 leads to an increase in the levels of Bif-1 and its phosphorylated form upon EBSS starvation. In conclusion, the present study for the first time reports HAX1 interacts with Bif-1 and regulates autophagic cell death upon EBSS starvation HAX1: Hematopoietic Cell Specific Lineage substrate 1 associated protein X 1 Bif-1: BAX interacting Factor 1 EBSS: Earle’s Balanced Salt solution 5 Abbreviations °C Degree Celsius µg Microgram mg Milligram ml Milliliter mM Millimolar M Molar L Liter µl Microliter sec Second µM Micromolar hr Hour min Minute mA Milliampere V Volt RT Room temperature Rpm Rotation per minute APS Ammonium peroxodisulfate TEMED N, N, N,-Tetramethylethylenediamine EDTA Ethylendiamine tetraacetate EGTA Ethylenglycol tetraacetate Tris Tris (hydroxymethyl)-aminoethane IPTG Isopropyl b-D-1-thiogalactopyranoside DTT Dithiothreitol DMSO Dimethyl sulphoxide DMF Dimethyl Formamide PMSF Phenylmethylsulphonyl fluoride FPLC Fast performance liquid chromatography GFP Green fluorescent protein HRP Horse radish peroxidase 6 LB Luria broth TBST Tris buffer saline Tween-20 PBS Phosphate buffer saline TAE Tris acetate EDTA SDS Sodium dodecyl sulfate Yeast abbreviation AD/library a fusion of GAL4 AD with library cDNA/ protein DNA- BD/ bait A fusion of the GAL4-BD with bait protein Yeast phenotypes Ade-, His-, Leu-, or Trp- requires adenine, histidine, leucine or tryptophan in the medium to grow; is auxotrophic for at least one of these specific nutrients Miscellaneous Ade2p Protein encoded by the yeast ADE2 gene 3-AT 3-amino-1, 2, 4-triazole; a competitive inhibitor of the His3 protein DO Dropout ( supplement or solution); a mixture of specific amino acids and nucleosides used to supplement SD base to make SD medium; DO solutions are missing one or more of the nutrients required by untransformed yeast to grow on SD medium. His3p protein encoded by the yeast His3 gene SD medium Minimal Synthetic Dropout medium; comprised of a nitrogen base, a carbon source (glucose or galactose) and a DO supplement. Bif-1 BAX interacting factor 1 UVRAG Ultraviolet Irradiation resistance-associated gene PI(3)KC(3) Class III Phosphatidylinositol 3-Kinase 7 Table of Contents 1. Introduction 14 1.1 Cancer 14 1.2 Cell death 14 1.3 TYPE 1 PROGRAMMED CELL DEATH (APOPTOSIS) 15 1.4 Extrinsic and Intrinsic pathways 17 1.5 TYPE II PROGRAMMED CELL DEATH (AUTOPHAGY) 19 1.5.1 mTOR complexes 21 1.5.2 Regulation of Autophagy by TOR 22 1.5.3 Role of Beclin1 in Autophagy 24 1.6 HAX1 26 1.7 HAX1 Protein Domains 29 1.8 HAX1 and its Biological role 30 1.8.1 Interaction with Viral Encoded Proteins 30 1.8.2 HAX1 as an Anti Apoptotic Molecule 31 1.8.3 HAX1 and its regulatory role in cell migration 32 1.8.4 HAX1 interaction with mRNA 34 1.8.5 HAX1 and its role in human diseases 34 1.9 Aims of the proposed study 37 2. Materials and Methods 38 2.1 MATERIALS 38 2.1.1CHEMICALS 38 2.1.2 Kits 40 2.1.3 Antibiotics 40 2.1.4 Resins for purification 41 2.1.5 Instruments 41 8 2.1.6 Cloning and Expression Vectors 41 2.1.6.1 Subcloning and Gateway cloning entry vectors 41 2.1.6.2 E.coli cloning vectors 42 2.1.6.3 Yeast cloning vectors 42 2.1.6.4 Dictyostelium discoideum cloning vectors 42 2.1.6.5 Mammalian cloning vectors 42 2.1.6.6 Retroviral cloning vectors 43 2.1.6.7 E.coli expression constructs 43 2.1.6.8 Mammalian system Expression constructs 43 2.1.6.9 Deletion mutants of H HAX1 44 2.1.6.10 Yeast Expression constructs 44 2.1.6.11 Dictyostelium discoideum Expression constructs 44 2.1.6.12 Retroviral Expression constructs 45 2.1.7 List of oligonucleotides (5’ to 3’) 45 2.1.8 Enzymes for molecular biology 46 2.1.9 Antibodies 46 2.1.9.1 Primary Antibodies 46 2.1.9.2 Secondary Antibodies 46 2.1.10 Standards 47 2.1.10.1 Size of DNA molecular weight marker 47 2.1.10.2 Molecular weight marker for proteins 47 2.1.11 General Buffers and Stock solutions 47 2.1.12 Materials for the cultivation of Escherechia coli 48 2.1.12.1 E.coli strains 48 2.1.12.2 Media and Buffers for the cultivation of E.coli 48 2.1.13 Materials for culturing Yeast Saccharomyces cerevisiae 49 2.1.13.1 Media 49 2.1.13.2 Solutions for yeast transformation 49 2.1.13.3 Solutions for X-Gal Colony lift filter assay 50 2.1.14 Materials for the cultivation of D.discoideum 50 2.1.14.1 Media and Buffers 50 2.1.15 Mammalian cell culture system 51 9 2.1.15.1 Cell lines 51 2.1.15.2 Solutions required for cell culture 52 2.1.15.3 Reagents for the production of retroviruses 53 2.1.15.4 Reagents for retrovirus transduction 53 2.1.15.5 Reagents for Transient cell transfection 53 2.2 Methods 54 2.2.1 Molecular biological methods 54 2.2.1.1 Isolation of plasmid DNA 54 2.2.1.2 Enzymatic modification of DNA 54 2.2.1.3 Amplification of DNA 54 2.2.1.4 Preparation of RNA 54 2.2.1.5 First Strand cDNA sysnthesis 55 2.2.1.6 Electrophoresis of DNA and Extraction from Agarose gels 55 2.2.1.7 pGEMT cloning of PCR products 55 2.2.1.8 5’–dephosphorylation of DNA fragments 56 2.2.1.9 Ligation of DNA fragments 56 2.2.1.10 Gateway cloning 56 2.2.1.11 pENTR-D/TOPO entry cloning 56 2.2.1.12 LR recombination 57 2.2.1.13 Preparation of Chemically competent E.coli cells 57 2.2.1.14 Preparation of Electro competent bacteria 57 2.2.1.15 Transformation of bacteria 58 2.2.1.15.1 Heat shock protocol 58 2.2.1.15.2 Electroporation of bacteria 58 2.2.2 Yeast Two hybridization Screen 59 2.2.2.1 Principle 59 2.2.2.2 Yeast Transformation 59 2.2.2.3 Transactivation test 60 2.2.2.4 Protein expression 60 2.2.2.5 Library transformation-two hybrid screen 60 10 2.2.2.6 X-Gal colony Lift filter assay 61 2.2.2.7 Isolation of Yeast plasmids 61 2.2.3 Dictyostelium discoideum 62 2.2.3.1 Transformation D.discoideum cells 62 2.2.3.2 Cultivation of D.discoideum cells 63 2.2.3.3 Conservation of D.discoideum spores 63 2.2.3.4 Cryo-conservation of vegetative cells 64 2.2.4 Protein Biochemistry 64 2.2.4.1 Preparation of cell lysates for western blot analysis 65 2.2.4.2 Protease inhibitor cocktail 66 2.2.4.3 Determination of Protein concentration 66 2.2.4.4 Preparation of cell lysates for Immunoprecipitation 67 2.2.4.5 Preparation of D.discoideum protein lysate 67 2.2.4.6 SDS polyacrylamide gel electrophoresis 67 2.2.4.7 Immunoblotting 68 2.2.5 Mammalian cell splitting and transfection 69 2.2.5.1 Cell splitting 69 2.2.5.2 Cell maintenance 70 2.2.5.3 Cell transfection 70 2.2.5.4 Mammalian cell fixation 70 2.2.5.4.1 PFA fixation 70 2.2.5.5 Immunofluorescence microscopy 70 2.2.5.6 Confocal Microscopy 71 2.2.5.6.1 Colocalization Analysis 71 2.2.5.7 Cell proliferation and Viability by MTT 71 2.2.5.8 Generation of Retroviruses 71 2.2.5.9 Determination of Viral titer 72 3.
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