Proteomic characterization of novel protein protein interactions for understanding functions of gene products

Proteomic Characterization of Novel Protein-Protein Interactions for Understanding Functions of Gene Products Yeo Wee Ming B.Sc. (HONS) in Genetics, University College London, U.K. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 i ACKNOWLEDGEMENTS Ernest appreciation to the following people: My supervisor, A/P Vincent Chow, for his understanding, patience and constant guidance. Lecturers of the Department of Microbiology, especially Prof. Chan Soh Ha, A/P Sim Tiow Suan, A/P Poh Chit Laa, A/P Mary Ng and Dr. Wong Siew Heng for their advice and encouragements. All the staff of the Department of Microbiology, especially Lim Ek Wang, Phoon Meng Chee and Josephine Howe, for pushing me on when I needed it. All the staff and students in the Human Genome Laboratory for having gone through the ups and downs of the project with me. My parents for supporting me spiritually and financially before and during my embarkment on this research project. My parent-in-laws for their constant concerns and encouragements. Cathy and Emma for understanding when I cannot be around during dinner and playtime. ii CONTENTS Page TITLE i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x CHAPTER 1: ABSTRACT 11 CHAPTER 2: LITERATURE REVIEW 12 CHAPTER 3: MATERIALS AND METHODS 57 CHAPTER 4: RESULTS AND DISCUSSION 71 CHAPTER 5: CONCLUSION 106 CHAPTER 6: REFERENCES 121 APPENDIX 148 PUBLICATIONS 151 INTERNATIONAL CONFERENCES 152 iii CONTENTS Page • Chapter 1: Abstract 11 • Chapter 2: Literature Review 15 o 2.1 The Yeast Two-Hybrid System 16 o 2.2 Picornaviruses 21 ƒ 25 2.2.1 Enterovirus o 2.3 Herpesviridae ƒ 2.3.1 Betaherpesvirinae 2.3.1.1 Classification and Subtypes 30 31 32 Classification 32 Subtypes 32 Host-cell tropism 33 Receptor interactions 34 Permissive and latent infections by HHV-6 34 Effect on T-cells 36 2.3.1.2 Genetics and molecular biology 36 Genome Organization and Relationship to other Herpesvirus 36 Viral origin of DNA replication 40 2.3.1.3 Viral genes 42 Viral glycoproteins 43 Other viral genes, including the viral protease 44 2.3.1.4 Association with disease 47 Primary infection and association with exanthema iv Page subitum (roseola) 48 Association with neurological disease 49 Viral infection of oligodendrocytes and association with multiple sclerosis 50 Association with malignancies 52 2.3.1.5 Association with disease in immunocompromised individuals 52 Role in chronic fatigue immunodeficiency syndrome 53 2.3.1.6 Relationship between HHV-6 and HIV-1 54 2.3.1.7 Clinical implications and applications 55 Management of severe complications of infection, including neurological disease. • Chapter 3: Materials and Methods 56 57 ƒ 3.1 Enterovirus 71 58 ƒ 3.1.1 Bacterial and Yeast Transformations 58 ƒ 3.1.2 in vivo protein expression 59 ƒ 3.1.3 Yeast Two-hybrid techniques 60 ƒ 3.1.4 Culturing of mammalian cells 61 ƒ 3.1.5 in vivo transcription/translation assay 62 ƒ 3.1.6 Cellular transfection and translation assays 62 ƒ 3.1.7 Confocal immunofluorescent staining assay 63 ƒ 3.1.8 EV71 infection of VERO cells 64 v Page 3.2 Human Herpesvirus o 65 ƒ 3.2.1 Bacterial and Yeast Transformations 65 ƒ 3.2.2 in vivo protein expression 66 ƒ 3.2.3 Yeast mating experiment 66 ƒ 3.2.4 Propagation of the numan MT-4 cell line, transfection and coimmunoprecipitation assays ƒ 3.2.5 Immunofluorescnece confocal microscopy ƒ 3.2.6 Determination of virus titer by TCID50 and assay assay for cell viability ƒ 66 67 68 3.2.7 Short hairpin RNA (shRNA) constructs to induce specific antiviral response by RNA interference (RNAi) 68 ƒ 3.2.8 Real-time RT-PCR 69 ƒ 3.2.9 Transmission electron microscopy 70 ƒ 3.2.10 Mitochondrial membrane potential assay by flow cytometry Chapter 4: Results and Discussion 70 71 4.1 EV71 72 4.2 U95 88 • Chapter 5: Conclusion 106 • Chapter 6: References 121 • Appendix 148 vi LIST OF TABLES Page 1. Table 1: Analysis of the frequency of structural abnormalities Of mitochondria in HHV-6B–infected versus Uninfected MT-4 cells at 0, 1, and h post-infection 97 2. Table 2: Genetics and cell infection of human herpesvirus (HHV-6) and human herpesvirus (HHV-7) 110 3. Table 3: Genetics of human herpesvirus (HHV-6) and human herpesvirus (HHV-7) 112 4. Table 4: Primers designed for use specifically in the Yeast Two-Hybrid experiments 119 5. Table 5: Primers designed for VP1 and U95 in the Yeast Two-Hybrid experiments 120 vii LIST OF FIGURES Figures Page 1. 2.1.1: Principle of the Two-Hybrid system 17 2. 2.2.1: Cartoon representation of a typical life cycle of Picornavirus 23 3. 2.2.2: Cartoon representation of the assembly and packaging of a virion 24 4. 2.2.1.1:Genotypic classification of Enterovirus 71 27 5. 2.2.1.2 Structure of EV71 genome 28 6. 2.2.1.3 Typical route of infection, spread and egression of an enterovirus 29 7. 2.3.1.2.1Genome organization of human herpesvirus (HHV-6) and human herpesvirus (HHV-7) 7. 2.3.1.2.2 Schematic diagram of human herpesvirus oriLyt 39 41 8. 2.3.1.3.1 Alignment of the protein sequences of the serine proteases of human cytomegalovirus (HCMV), human herpesvirus and human herpesvirus 46 9. 4.1.1: Gel electrophoresis photographs of colony PCR 74 10. 4.1.2: β-galactosidase assay 74 11. 4.1.3: The result for the co-immunoprecipitation of VP1 and Ornithine Decarboxylase 12. 4.1.4: The result for the co-immunoprecipitation of VP1 and GTAR 75 76 13. 4.1.5: The result for the co-immunoprecipitation of VP1 and KIAA0697 77 14. 4.1.6: Co-localization of EV71 VP1 with ODC1 78 15. 4.1.7: Co-localization of EV71 VP1 with GTAR 79 viii Figures 16. 4.1.8: Co-localization of EV71 VP1 with KIAA0697 Page 80 17. 4.1.9: Specific detection of wild-type VP1 protein in EV71-infected cells 81 18. 4.1.10: Kinetics of co-localization of wild-type VP1 protein with endogenous ODC1 in EV71-infected cells 19. 4.1.11: Negative controls for immunofluorescence studies 82 83 20. 4.2.1: Co-immunoprecipitation of HHV-6B U95 and human GRIM-19 proteins in MT-4 cells 21. 4.2.2: Intracellular co-localization of HHV-6B U95 with GRIM-19 89 91 22. 4.2.3: Co-localization of HHV-6B U95 with endogenous GRIM-19 in MT-4 cells 92 23. 4.2.4: Real-time RT-PCR analyses of relative U95 mRNA expression levels in HHV-6B-infected MT-4 cells following U95-shRNA treatment compared with controls 93 24. 4.2.5: Transmission electron micrographs of HHV-6B-infected cells without and with U95-shRNA treatment 96 25. 4.2.6: Assessment of mitochondrial membrane potential by the MitoProbe DiOC2(3) assay 100 ix ABBREVIATIONS aa BCIP bp CA16 cDNA c-Myc CPE DNA dNTP FITC FLAG HA HFMD kb KDa lacZ LB LiAc M mg mins ml mM MOI NBT ng nt OD ORF PAGE PBS PCR RNA RNase SDS TBST X-gal YPD amino acids 5-bromo-4-chloro-3-indoyl phophate base pairs Coxsackievirus 16 complimentary DNA Human oncoprotein Cytopathic effect Deoxyribonucleic Acid Deoxyribonucleotide triphosphate Fluorescein isothiocyanate Short DYKDDDDK polypeptide Short YPY DVP DYA polypeptide from hemagglutinin influenza virus Hand, foot and mouth disease Kilobase Kilodalton Z gene of lactose operon (encoding the enzyme βgalactosidaes Luria Broth Lithium acetate Molar Milligram minutes Millilitre Millimolar Multiplicity of infection Nitroblue tetrazolium Nanogram Nucleotide Optical density Open reading frame Polyacrylamide gel electrophoresis Phosphate-buffered saline Polymerase chain reaction Ribonucleic Acid Ribonuclease Sodium dodecyl sulphate Tris-borate-EDTA Tween 20 5-bromo-4-chloro-3-indoyl-β-galactopyranoside Yeast peptone dextrose x oxidative processes and of myelin figures formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7ketocholesterol-induced apoptosis: comparison with various pro-apoptotic chemicals. 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Lancet. 1: 1065-7. 217. Yeo, W. M., and V. T. Chow. 2007. The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat. Microb. Pathog. 42: 129-137. 147 APPENDIX 1. LB medium (LB) Bacto-Tryptone 10 grams Bacto-yeast extract grams NaCl 10 grams ddH2O to litre Note: adjust pH to 7.0 and autoclave to sterilize 2. Dulbecco modified eagle’s minimum essential medium with 10% fetal calf serum (DMEM10) Dulbecco modified eagle’s medium (Gibco) Packet HEPES (Gibco) 2% NaHCO3 2% of 7% stock FCS 10% The solution was then adjusted to 1L and filtered through a 0.22µm syringe filter. 3. RPMI (1640) 10 RPMI (1640) (Gibco) Packet HEPES (Gibco) 2% NaHCO3 2% of 7% stock FCS 10% L-glutamine 3g The solution was then adjusted to 1L and filtered through a 0.22µm syringe filter. 4. Tris-EDTA buffer Tris-HCl 10mM EDTA 1mM 148 5. SDS Loading buffer 1M Tris, pH 6.8 625µl 10% SDS 2ml 2-β-mercaptoethanol 0.5ml Glycerol 1ml Bromophenol Blue 0.003g Top up with double distilled water to 10ml. 6. ESB solution SDS 2% Tris pH6.8 80mM Glycerol 10% DTT 1.5% Bromophenol Blue 0.1mg/ml 7. YPDA/Kan Yeast extract 1% Peptone 2% Dextrose 2% Adenine hemisulphate 0.003% Kanamycin 15mg/L The kanamycin and adenine hemisulphate are filtered sterilized and added to the autoclaved solution only after cooling it to below 550C. 8. PEG/LiAc PEG 3350 40% Tris-EDTA 1X 149 Lithium Acetate 1X 9. Z buffer (pH7.0) Na2HPO4.7H20 16.1g/L NaH2PO4.H2O 5.50g/L KCl 075g/L MgSO4.7H2O 0.246g/L 10. X-Gal stock solution 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was dissolved in DMF (Sigma-Aldrich) to reach a final concentration of 20mg/ml. 10. Z buffer / X-Gal solution Z buffer 100ml Β-mercaptoethanol 270µl X-gal stock solution 1.67ml 11. Fluorescent Dilution Buffer (FDB) MgCl2 1mM CaCl2 1mM Normal porcine serum 5% Fetal calf serum 5% Bovine serum albumin 2% PBS, pH 7.4 150 PUBLICATIONS 1. Yeo WM, Isegawa Y, Chow VT. 2008. The U95 protein of human herpesvirus 6B interacts with human GRIM-19: silencing of U95 expression reduces viral load and abrogates loss of mitochondrial membrane potential. J. Virol. 82(2):1011-20. 2. Yeo WM, Chow VT. 2007. The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat. Microb. Pathog. 42(4):129-37. 151 INTERNATIONAL CONFERENCES 1. Yeo W. M. and V. T. K. Chow, “Interactions of the VP1 structural protein of enterovirus 71 (EV71) with human ornithine decarboxylase and gene trap ankyrin repeat provide insights into EV71 pathogenesis”. 8th NUS-NUH Annual Scientific Meeting, comp. T. S. Sim, B. H. Bay (2004): 52. Singapore: 8th NUS-NUH Annual Scientific Meeting, 7-8 October 2004. Publication number 0204932. 2. Chow, V. T. K., W. M. Yeo and Y. Isegawa, “Interaction of the U95 protein of human herpesvirus with the GRIM19 cell death regulatory protein”. Genomic Med. and Population Health, comp. Liu E. (2004): 152. Singapore: Human Genome Organization. (5th Human Genome Organization (HUGO) Asia Pacific Meeting and 6th Asia Pacific Conference on Human Geneics, 17-22 November 2004). Publication number 0204992. 152 [...]... screening tool for the identification and isolation of potential interacting protein partners for your protein of interest The knowledge requirement for your protein of interest is only very minimal If a protein has a known function, new proteins that bind to it bring additional components into play, ultimately contributing to understanding the process under study Alternatively, the function of a protein. .. two-hybrid system also allows for the analysis of known interactions Fourthly, the identification of an interacting protein implies that at the same time the corresponding gene is cloned Last but not least, modification of this system has allowed for studies of protein- DNA and protein- RNA interactions Knowing the abilities of the system will not allow one to maximize the capabilities of the system unless the... metabolism, formation of cellular macrostructures and enzymatic complexes Hence to understand every individual functions of a single protein, we need to look at the participation of the protein in all the biological pathways; interacting proteins might give a functional hint if at least one of the partners has a known functional commitment in a well understood biological pathway The study of protein- protein interactions. .. encephalitis To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection To study the functional role of the HHV-6B IE gene, U95, we generated a U95-Myc fusion protein, and screened a pre-transformed bone marrow cDNA library for U95-interacting proteins using yeast-two hybrid analysis... than those of mammals, which necessitates that its cellular context differs from that of a mammalian cell The latest development involving looking at protein- protein interactions in the mammalian cells, the two-hybrid systems in mammalian cells, can ensure that the protein protein interactions under scrutiny happen in a more 'natural' environment When it comes to studying protein protein interactions. .. yeast genetic assay in which the interaction of two proteins is measured by the reconstitution of a functional transcription activator in vivo (Fields and Song, 1989, Chien et al, 1991) With this method, mutational analysis can also be done to pin-point the exact location of the domains/residues necessary for the interaction Secondly, only the cDNA, full-length or even partial of the 18 gene of interest... as Candida albicans can cause infection in humans More than one-thousand species of yeasts have been described The most commonly used yeast is Saccharomyces cerevisiae, which was used for wine, bread and beer production thousands of years ago The yeast two-hybrid system is a novel way for detection of protein- protein interactions in-vivo in Saccharomyces cerevisiae (baker’s yeast) As Saccharomyces... three major domains: identification, characterization and manipulation Traditionally, the tools available to analyze proteinprotein interactions in multicellular organisms have been restricted to biochemical approaches in vitro Biochemical approaches can be time-consuming and that detection of proteins that bind to another proteins generally result in the appearance of a band on an immobilized matrix... polypeptides necessary for replication The 5’ untranslated region (UTR) contains the determinants for translation of the viral RNA by internal ribosomal entry site (IRES) mechanism for amplification of viral RNA and for neurovirulence (Evans et al, 1985) The 3’ UTR region contains a pseudo-knot like structure that is important for replication of viral RNA High-resolution structures of rhinoviruses (Rossmann... been solved by x-ray diffraction The viral capsid of picornaviruses consists of a densely packed icosahedral arrangement of 60 protomers, composed of four different proteins (VP1-VP4) each, and the shell is arranged on a pseudo T 5 3 symmetry (p 5 3) with a diameter of 27–30 nm The protein subunits are produced from a proteolytic cleavage of the polyprotein (Oliveira et al, 1999) These subunits are . i Proteomic Characterization of Novel Protein- Protein Interactions for Understanding Functions of Gene Products Yeo Wee Ming B.Sc. (HONS) in Genetics, University. valuable screening tool for the identification and isolation of potential interacting protein partners for your protein of interest. The knowledge requirement for your protein of interest is only. study the functional role of the HHV-6B IE gene, U95, we generated a U95-Myc fusion protein, and screened a pre-transformed bone marrow cDNA library for U95-interacting proteins using yeast-two