Efficacy of Mutant HPV-16 E6 Proteins in p53 Degradation

Background and Objective:   High-risk human papilloma viruses (HPV) are the causative agent in the majority of anal, cervical, vaginal, vulvar, penile, and oropharyngeal cancers with an annual incidence of 630,000 cases world-wide. These viruses initially cause dysplasia that subsequently increases neoplasia risk. HPV’s encode eight major viral proteins with the E6 protein being crucial for replication. E6 binding to ubiquitin ligase E6AP initiates polyubiquitination of p53, targeting the protein for proteasomal degradation. We are taking a novel approach to inhibit HPV16+ dysplasia and cancers by designing inhibitors targeting specific amino acids of 16E6, which reside in the E6-E6AP binding pocket, thereby preventing p53 degradation. To affirm interaction with the targeted side chain, we generated E6 point mutations that serve as specificity controls. These mutations are designed to disrupt interaction with the compound. To ensure their suitability for our studies, we are herein characterizing their capacity to bind E6AP and bind and degrade p53.     Methods:   E6 mutants were generated by site-directed mutagenesis and analyzed by sequencing. H1299 cells were transfected with GFP, wild-type (WT) E6, or mutant HPV-16 E6 plasmids +/- WT p53 plasmid. After 48 hours, cells are lysed and 16E6 was immunoprecipitated. Proteins bound to 16 E6 were separated by SDS-PAGE and subjected to western blot. Binding to E6AP and p53 was analyzed and presence of 16E6 was confirmed by immunoblotting. To test for p53 degradation, H1299 cells were transfected with firefly luciferase (Fluc) (transfection control) and p53-luciferase along with WT E6, mutant E6 proteins, or empty LXSN plasmid. After 24 hours p53-luciferase was measured with Dual-Glo Luciferase Assay.     Results:    To be determined    Conclusion:     To be determined