Analysis of the HPV E6 "proteome", "ubiquitome" and "interactome"

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TRAUSCH, Myriam, 2013. Analysis of the HPV E6 "proteome", "ubiquitome" and "interactome" [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Trausch2013Analy-27558, title={Analysis of the HPV E6 "proteome", "ubiquitome" and "interactome"}, year={2013}, author={Trausch, Myriam}, address={Konstanz}, school={Universität Konstanz} }

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect cutaneous and mucosal epithelial cells. Mucosal HPVs can be divided in high risk and low risk types depending on their association with clinical lesions. Infection with high risk types can lead to the development of cervical cancer, whereas infection with low risk types induces the formation of benign genital tumors. E6 proteins of high risk HPVs contribute to the development of cervical cancer by utilizing the cellular ubiquitin ligase E6AP to target the tumor suppressor p53 and several other cellular proteins for degradation. In contrast, E6 proteins of low risk HPVs are only weakly oncogenic and only little is known about their biochemical and physiological properties.<br /><br /> In order to obtain further insights into the role of the high risk and low risk E6 proteins in the development of cervical cancer and benign tumors, in the first part of this thesis, the HPV E6 proteins were analyzed with respect to their influences on the “proteome” and “ubiquitome” of cells. In addition, interaction partners of HPV E6 were identified. To determine changes on proteomes upon expression of E6 proteins (“HPV E6 proteome”), we developed an inducible expression system that allows switching on and off E6 expression within cells. We used this system combined with quantitative mass spectrometry (stable isotope labeling of amino acids in cell culture, SILAC) and determined the effect of different E6 proteins on the protein expression pattern of a cell in general. For the identification of proteins that are ubiquitylated in an E6-dependent manner (“HPV E6 ubiquitome”), an in cellulo assay using tandem ubiquitin binding entities (TUBEs) as stabilization and isolation tool of ubiquitylated proteins was established. For the “interactome” studies (proteins that interact with E6), pulldown experiments with GST-tagged E6 and cell lysates from cells that do or do not express E6AP in combination with quantitative mass spectrometry (SILAC) were performed. With this approach, three subunits of the Anaphase Promoting Complex (APC/C) among others were identified as interaction partners of E6. APC/C is an E3 ligase whose major role is controlling the cell cycle. The interaction between APC/C and the HPV E6 proteins could be verified. However, further investigations are necessary to elucidate the physiological function of this interaction. In conclusion, this study contributes to the elucidation of the cellular pathways that are affected by both low risk and high risk E6 proteins.<br /><br /> In the second part of this thesis we showed that the E6 interacting protein LNX1 is able to bind to the tumor suppressor p53 and that it induces ubiquitylation and degradation of p53 in an E6-independent manner. p53 is a tightly regulated protein and has critical functions including regulation of cell death, senescence and proliferation. Our data show that an additional E3 ligase, LNX1 is involved in the regulation of p53. Analysis of the HPV E6 "proteome", "ubiquitome" and "interactome" Trausch, Myriam 2014-04-11T08:30:45Z Trausch, Myriam 2013 terms-of-use eng

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