Novel biochemical and functional properties of the HPV 16 oncoprotein e6

NOVEL BIOCHEMICAL AND FUNCTIONAL PROPERTIES OF THE HPV16 ONCOPROTEIN E6 ROLAND DEGENKOLBE (M.Sc. (chemistry), University of Cologne) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2003 for me mum and me dad II Acknowledgements First and foremost I would like to thank my supervisor Hans-Ulrich Bernard for his unwavering support and guidance throughout all the years I spent in his laboratory. A big thank you goes to Holger Zimmermann and Mark O’Connor for the advice, the help, the discussions and for giving me the chance to contribute to (and profit from) their work. Thank you! Many thanks to Walter Stünkel and Tan Shih-Han for discussions and advice. More thanks to: John McCarty for giving me all the essential advice about protein purification and, occasionally, cheese-cakes. Patrick Clement Gilligan for inspiring conversations and the (only partially successful) degermanization of my writing (and thinking). Sanjay Gupta for an auspicious collaboration and many laughs in the lab. Sushma Badal for an unending supply of coins keeping me fed and healthy. Param Parkash Singh Takhar (one person) for technical support and so many wuffs in the lab. Thank You, Eyleen. I would also like to thank (in no particular order): Apple (great hard- and software), honda (hardware), homer (in memoriam), at-the-drive-in, KTM, tool, monster magnet, chikako, sunnydale real estate, sonic youth, tim & honey (bad III influences), michael, haruki murakami, yuki (doreen that is; and yesyou’rerightandI’mwrong), IPA, the exploited, jarkko, snapcase, kumiko, the kfc at ginza plaza, pj harvey, toothbrush, NOFX, handlebar, the johor circuit, the economist-style guide, you know who (for showing again and again that obvious things can be made complex, nothing should be taken for granted, there’s always more brand-new rules than you thought there could be and never forget that you must not question), kemistry and storm, takagi, unity trading, the deftones, asiapacific-brewery (kept them firmly in the black), insomnia and, of course, all my …… IV Table of contents Acknowledgements III Table of contents .V List of figures .VIII Abbreviations and Acronyms X - SUMMARY 13 - INTRODUCTION . 14 2.1 Classification and history 14 2.2 Cervical cancer . 15 2.3 Virion structure 17 2.4 Genome organization . 18 2.5 The early proteins . 19 2.5.1 The E1 protein 19 2.5.2 The E2 protein 20 2.5.3 The E4 protein 20 2.5.4 The E5 protein 21 2.5.5 The E7 protein 21 2.5.6 The E6 protein 25 2.6 The E6 protein as a drug target 30 - RESULTS (PART 1): BIOPHYSICAL PROPERTIES OF E6 AND E7 . 33 3.1 Biophysical properties of the E6 and E7 proteins: Previous data and concept of this study. . 33 3.2 Biophysical properties of the E7 protein: Experimental data . 35 3.3 Biophysical properties of the E7 protein: Conclusion 42 3.4 Biophysical properties of the E6 protein: Previous reports and considerations for this study. . 43 3.5 Biophysical properties of the E6 protein: Experimental data . 45 3.6 Biophysical properties of the E6 protein: Discussion . 57 - RESULTS (PART 2): BIOLOGICAL PROPERTIES OF E6 60 V 4.1 Interaction of E6 with CBP/p300: Previous publications and considerations for this study 60 4.2 Interaction of E6 with CBP/p300: Experimental data 63 4.3 Interaction of E6 with CBP/p300: Discussion 76 - CONCLUSION 81 5.1 Concluding remarks . 86 - EXPERIMENTAL PROCEDURES . 88 6.1 Bacterial culture . 88 6.1.1 Growth of bacteria in liquid or solid media . 88 6.1.2 Preparation of competent cells 90 6.1.3 Transformation of competent cells 91 6.2 DNA 92 6.2.1 Quantitation of DNA and RNA 93 6.2.2 DNA amplification and purification 93 6.2.3 Gene assembly 96 6.2.4 Cloning of DNA fragments 98 6.2.4.1 Separation of DNA fragments with agarose gels .98 6.2.4.2 Restriction digest .99 6.2.4.3 Ligation . 100 6.2.4 Preparation of plasmid DNA .100 6.2.5 Site-directed mutagenesis 103 6.3 Protein .105 6.3.1 Expression of recombinant protein .105 6.3.2 Uniform labeling of protein .106 6.3.3 Lysis of bacteria .108 6.3.4 Metal affinity chromatography 109 6.3.5 Anionexchange chromatography .110 6.3.6 Size-exclusion chromatography .111 6.3.7 Hydroxyl-apatite chromatography 112 6.3.8 Dialysis .113 6.3.9 Renaturation (for E7 only) 113 VI 6.3.10 Protein quantitation .114 6.3.11 Discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis 115 6.3.12 Zinc (II)-ion quantitation .119 6.3.12.1 TSQ-assay . 119 6.3.12.2 Determination of zinc content by Inductively Coupled Plasma/Optical Emission Spectroscopy (ICP/OES) 120 6.4 Protein-protein interaction assay with GST- “micro-columns” .121 6.5 in vitro p53 degradation assay .123 6.6 in vivo p53 degradation assay 123 - REFERENCES .125 VII List of figures Figure 1: Prevalence of different cancers in cancer related deaths in women. Figure 2: Picture of the capsid of human papillomavirus. Figure 3: Organization of the genome of HPV16. Figure 4: Schematic diagram of the HPV16 E7 protein. Figure 5: Schematic diagram of the HPV16 E6 protein. Figure 6: Expression and metal-affinity purification of HPV16 E7. Figure 7: Apparent size distribution of HPV16 E7. Figure 8: No evidence for covalently linked dimers or multimers of HPV16 E7. Figure 9: The influence of chelating agents on the agglomeration of E7. Figure 10: Agglomeration of renatured E7 protein is pH dependent. Figure 11: Solubility of S-E6 after expression in E.coli is pH dependent. Figure 12: Purification of S-E6 expressed in E. coli. Figure 13: Apparent size distribution of purified S-E6 (after anionexchange chromatography and dialysis). Figure 14: Zinc(II) ions interfere with binding of E6 to an E6AP peptide. Figure 15: Differences in the proportion of multimeric to monomeric S-E6 after dialysis in the presence of a small variety of chelating agents. Figure 16: Competition for zinc by three different chelating agents. Figure 17: Monomeric S-E6 is biologically more active compared with its multimeric form in catalysis of p53 degradation. Figure 18: Agglomerated protein is destabilized by addition of a chelating agent. VIII Figure 19: Medium-scale preparation of S-E6. Figure 20: HPV16 E6 interacts with the transcriptional coactivator CBP/p300. Figure 21: Identification of an HPV16 E6 binding site on CBP/p300. Figure 22: Interactions of HPV16 E6 with the cellular protein p300. Figure 23: The E6-CBP/p300 interaction is specific for E6 proteins of highrisk HPVs. Figure 24: Mapping of the E6 domain interacting with CBP. Figure 25: The 16E6 mutant L50G binds CBP but is unable to interact with E6AP or p53 and cannot degrade p53 in vitro or in vivo. Figure 26: Amino acid sequence of HPV16 E6 and predicted secondary structure. Figure 27: Model of combinatorial binding modes of domains of E6 Table 1: Stoichiometric ratios of zinc to S-E6 after individual steps of purification and dialysis with different chelating agents. IX Abbreviations and Acronyms µ micro, 10-6 a atto, 10-18 AP alkaline phosphatase ATP adenosine triphosphate ATCC American Type Culture Collection AU absorbance units ßME beta-mercapto-ethanol bp base pair BSA bovine serum albumin CD circular dichroism cpm counts per minute CTP cytosine triphosphate Da Dalton, atomic mass unit, 1.67377 x 10 –27 kg ddH2O double-destilled water DMSO dimethyl sulfoxide DNase deoxyribonuclease dNTP deoxynucleoside triphosphate DTT dithiothreitol EDTA ethylenediamine- N,N,N’,N’-tetraacetic acid EGTA ethyleneglycol-bis-(b-aminoethyl)-N,N,N’,N’-tetraacetic acid f femto, 10-15 X – EXPERIMENTAL PROCEDURES 124 h after cycloheximide treatment and lysed. 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[...]... of about 8kbp 2.4 Genome organization The circular HPV1 6 genome has eight ORFs, encoding six early genes (E1, E2, E4, E5, E6 and E7) and two late genes (L1 and L2), and a long control region (LCR) located between the L1 and E6 ORFs (Figure 3) Figure 3: Organization of the genome of HPV 16 Most of the ORFs overlap and the early genes are all transcribed from a single promoter, P97, upstream of the E6. .. 2.5.6 The E6 protein The E6 protein of HPV1 6 is a small polypeptide of 151 amino acids and contains two putative zinc binding motifs (Barbosa, Lowy, and Schiller, 1989; Cole and Danos, 1987), as illustrated in figure 5, which are crucial for all but a few of the numerous different functions of E6 (Kanda et al., 1991; Sherman and Schlegel, 1996) The first pieces of evidence that E6 is a viral oncoprotein. .. incapacitated otherwise but are being continuously expressed and then degraded, a loss of the function of E6 or E7 would be catastrophic for the cancer cell The presence of functional pRB and p53 in a late cancer cell would immediately arrest the cell-cycle and most likely initiate apoptosis (Francis, Schmid, and Howley, 2000) This continuous dependency of the cancer cell on E6 or E7 for survival, the lack of. .. Biophysical properties of E6 and E7 3.1 Biophysical properties of the E6 and E7 proteins: Previous data and concept of this study To determine the biophysical properties of proteins and, ultimately, determine their three-dimensional structure (a prerequisite for rational drug-design), one needs native, pure, concentrated and homogenous protein The two methods employed routinely for determination of high-resolution... expression of the cyclins E and A (Hickman, Picksley, and Vousden, 1994) and cdc25A (Katich, Zerfass-Thome, and Hoffmann, 2001), inactivation of the p53-responsive CKI p21CIP1 (Funk et al., 1997; Jones, Alani, and Munger, 1997), and the decreased steady-state levels of pRB (Jones and Munger, 1997) Cells expressing E7 show increased levels of p53 (Demers, Halbert, and Galloway, 1994) and the normal... degradation of p53 mediated by the cellular ubiquitin ligase MDM2 seems to be disturbed (Jones and Munger, 1997; Seavey et al., 1999) Nonetheless, the rapid turnover of p53, a prerequisite for cell immortalization, in HPV positive cells, expressing both, E7 and E6, is entirely due to one of the main functions of the E6 gene product, the E6AP dependent targeting of p53 for ubiquitin dependent degradation via the. .. by a progressive spectrum of abnormalities of the cervical epithelium (Richart and Barron, 1969) These lesions are classified as cervical intraepithelial neoplasia (CIN) grades 1, 2 and 3 The severity of the lesion is graded by the extent to which the normally differentiating, non-mitotic suprabasal cells of the cervical epithelium are replaced by the non-differentiating and mitotically active basal-like... for human papillomavirus (HPV) DNA, with about 60% of them positive for HPV1 6 Two of the eight known genes encoded by HPV1 6, the early genes 6 (E6) and 7 (E7) are responsible for cell-transformation and transition to malignancy The E6 protein binds to a cellular E3-ubiquitin ligase (E6AP) and this complex targets p53, an important cellular apoptosis messenger, for degradation E6 is expressed throughout... E6 or E7 for survival, the lack of a beneficial role for both and the lack of homologous cellular proteins make E6 and E7 excellent drug-targets An example of anti -HPV E6 and E7 drug development is a recent study targeting the cysteines of the two zinc fingers of E6 with oxidizing agents rendering them incapable of coordinating zinc and the protein useless to execute its transforming functions (Beerheide... HPV1 6 E7 protein; the conserved regions 1 and 2 (CR I, CR II; similar to regions conserved in Adenovirus E1A or SV40 large T), conserved region 3 (CR III), the pRB binding domain, the site for phosphorylation by the casein kinase II and the location of the zinc binding domain are indicated The HPV E6 protein consists of two tandem copies of this domain and it has been speculated that E6 and E7 may have . NOVEL BIOCHEMICAL AND FUNCTIONAL PROPERTIES OF THE HPV1 6 ONCOPROTEIN E6 ROLAND DEGENKOLBE (M.Sc. (chemistry), University of Cologne) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE. diagram of the HPV1 6 E7 protein. Figure 5: Schematic diagram of the HPV1 6 E6 protein. Figure 6: Expression and metal-affinity purification of HPV1 6 E7. Figure 7: Apparent size distribution of HPV1 6. (PART 1): BIOPHYSICAL PROPERTIES OF E6 AND E7 33 3.1 Biophysical properties of the E6 and E7 proteins: Previous data and concept of this study. 33 3.2 Biophysical properties of the E7 protein: Experimental