The significance of human papillomavirus E6 PDZ binding motif on the transformation and immortalization of human neonatal keratinocytes

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dc.contributor.authorDust, Kerry Ann Nichol
dc.contributor.examiningcommitteeCoombs, Kevin (Medical Microbiology and Infectious Diseases) Mark, Brian (Microbiology) Archambault, Jacques (McGill University)en_US
dc.contributor.supervisorSeverini, Alberto (Medical Microbiology and Infectious Diseases)en_US
dc.date.accessioned2019-09-25T15:51:56Z
dc.date.available2019-09-25T15:51:56Z
dc.date.issued2019-09-10en_US
dc.date.submitted2019-09-18T19:41:37Zen
dc.degree.disciplineMedical Microbiology and Infectious Diseasesen_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US
dc.description.abstractBackground: All high-risk HPV have a PDZ protein-binding motif on the carboxyl-termini of their E6 proteins. This molecular feature is absent from low-risk HPV E6 proteins. The E6 PDZ binding motif permits interaction with PDZ proteins, including those of the tight junction and polarity complexes, leading to their degradation or mislocalization. Previously, the PDZ binding motif was found to enhance epithelial to mesenchymal transition of cells, an event marked by loss of tight junctions and polarity. The significance of the E6 PDZ binding motif for the immortalization and transformation of keratinocytes has not been elucidated, however, PDZ proteins are known to be targeted by other oncogenic viruses and are dysregulated in several epithelial cancers. Methods: Lentiviruses encoding the full-length E6 gene or a truncated E6 gene lacking the PDZ binding motif, in addition to lentiviruses encoding the E7 gene, were used to transduce keratinocytes. Longevity and growth rates of these experimental cells were assessed. Additionally, the expression of the pro-apoptotic protein, p53, and the immortalization–associated protein, hTERT were evaluated by Western blot. Quantitative LC/LC-MS/MS, coupled with pathway analysis software, examined the global protein dysregulation associated with the presence of the HPV16 E6 PDZ binding motif. Immunofluorescence microscopy was used to observe the level and localization of tight junction proteins. Results: Regardless of whether full-length or truncated E6 genes were expressed, the cells displayed increased longevity compared to control cells, indicating that the PDZ binding motif was not crucial for extending the proliferative capacity of keratinocytes. The E6 PDZ binding motif was associated with lower levels of p53 and increased levels of hTERT, which is significant as this demonstrates that the E6 PDZ binding motif enhances transformation-associated traits. E6 PDZ binding motif was not sufficient to induce anchorage-independent growth in soft agar. The level and location of PDZ proteins were altered in the cells expressing full length HPV16 E6 and E7 compared to the other cell lines. Pathway analyses predicted that cellular organization and immune responses are influenced by the E6 PDZ binding motif. Conclusions: The HPV16 E6 PDZ binding motif is not essential for immortalization of keratinocytes but does enhance transformation-associated traits including the degradation of p53 and expression of hTERT. The E6 PDZ binding motif also alters proteins associated with cellular organization and immune signaling pathways. HPV16 E6 and E7 were not sufficient to cause transformation of primary keratinocytes.en_US
dc.description.noteFebruary 2020en_US
dc.identifier.urihttp://hdl.handle.net/1993/34308
dc.language.isoengen_US
dc.rightsopen accessen_US
dc.subjectHuman Papillomavirusen_US
dc.subjectE6en_US
dc.subjectE7en_US
dc.subjecttransformationen_US
dc.subjectImmortalizationen_US
dc.titleThe significance of human papillomavirus E6 PDZ binding motif on the transformation and immortalization of human neonatal keratinocytesen_US
dc.typedoctoral thesisen_US
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