Biochemical characterization of the human papillomavirus type 16 E7 oncoprotein and its interaction with the retinoblastoma growth suppressor gene product
Abstract
Greater than 50% of all cervical carcinomas contain at least part of the human papillomavirus type 16 (HPV-16) genome. The HPV-16 E7 gene is necessary and sufficient for transformation of mammalian cells. This study was initiated to biochemically characterize the HPV-16 E7 oncoprotein and its interaction with the retinoblastoma growth suppressor protein (pRB) as a means toward understanding the mechanism of E7-mediated oncogenesis. This interaction is primarily mediated by the ten amino acid conserved region 2 (CR2) domain of E7. Recombinant E7 protein was expressed and purified from E. coli. An environmentally sensitive cysteine derivatization scheme was developed to assess the state of each cysteine residue, since five of the seven cysteine residues have been shown to be required for E7 function. The results of this study show that cysteine 24, located in the CR2 domain, is freely accessible to the solvent and available to interact with pRB. Cysteine residues 58, 61, 91 and 94, which comprise E7's conserved region 3 (CR3) domain, are likely to interact with a buried zinc ion, and contribute to structural stability. An E7 protein structure/function analysis was performed by generating multiple fragments of E7 and testing them in three E7-related assays. These data demonstrate that while the CR2 domain of E7 is required for tight binding to pRB, amino acids from the CR3 domain are also required for optimal activity in pRB/E7, pRB/E2F, and pRB/DNA binding inhibition assays. In fact, the CR2 domain alone is insufficient to inhibit pRB from binding to DNA or the transcription factor E2F, whereas the CR3 domain alone is sufficient to inhibit pRB from binding to E2F. The existence of an E7 CR3/pRB interaction was explicitly demonstrated by direct binding experiments utilizing recombinant pRB and a GST-E7 CR3 protein chimera. The observation that E7's CR3 domain displaces "active" E2F from the "inactive" pRB/E2F complex identifies a potential mechanism of E7-mediated growth stimulation and highlights the importance of the pRB/ E2F interaction in normal cells.
Subject Area
Biochemistry
Recommended Citation
Patrick, Denis R, "Biochemical characterization of the human papillomavirus type 16 E7 oncoprotein and its interaction with the retinoblastoma growth suppressor gene product" (1993). Dissertations available from ProQuest. AAI9413887.
https://repository.upenn.edu/dissertations/AAI9413887