| Specific subtypes of the human papillomavirus known as high-risk HPVs, especially subtypes 16 and 18, have been identified as the causative agents in the overwhelming majority of cervical cancers, of which there are 400,000 new cases annually. Genetic analysis has narrowed the oncogenic potential of HPV to two genes, E6 and E7, which encode proteins that bind and inactivate the tumor suppressor proteins p53 and pRb, respectively. E6 forms a complex with a ubiquitin-protein ligase known as E6-Associated Protein (E6AP), which ubiquitinates and targets p53 for degradation by the 26S proteasome. In this thesis, we present in vitro and in vivo evidence that the HPV16 E6 protein mediates degradation of E6AP by triggering a primarily intramolecular self-ubiquitination reaction. We have found a striking correlation between the ability of E6 to stimulate E6AP degradation and to immortalize human mammary epithelial cells. Importantly, the F2V, Y54H, and SAT8--10 mutants of E6 which retain immortalization activity but lose p53 degradation activity, also retain their ability to degrade E6AP. Finally, sequence analysis has revealed two PEST sequences within E6AP, one at the N-terminus (PEST1), the other (PEST2) in the center of the coding region spanning the E6-binding site. PEST sequences have been shown to be necessary and sufficient to target a number of other proteins for rapid degradation; both PEST sequences in E6AP also appear to act as independent degradation signals that can function in the absence or presence of the other. PEST2, but not PEST1, appears to require E6AP catalytic activity to act as a degradation signal; furthermore, PEST1 was able to function as a transferable degradation signal when fused to the normally stable green fluorescent protein (GFP). In summary, our efforts have focused on the degradation of the cellular ubiquitin-protein ligase E6AP, both in the presence and absence of the HPV E6 oncoprotein. It is hoped that the experiments presented will provide insights into both HPV-mediated oncogenesis and the function of the ubiquitin-proteasome pathway. |
