Scott Vande Pol

Papillomavirus E6 oncoproteins

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on these proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression.

Destabilization of TIP60 by human papillomavirus E6 results in attenuation of TIP60 dependent transcriptional regulation and apoptotic pathway

The TIP60 tumor suppressor is a histone acetyltransferase involved in transcriptional regulation, checkpoint activation, and p53-directed proapoptotic pathways. We report that human papillomavirus (HPV) E6 destabilizes TIP60 both in vivo and in vitro. TIP60 binds to the HPV major early promoter and acetylates histone H4 to recruit Brd4, a cellular repressor of HPV E6 expression. Both low- and high-risk HPV E6 destabilize TIP60, thereby derepressing their own promoter. Destabilization of TIP60 by HPV E6 also relieves cellular promoters from TIP60-initiated repression and abrogates p53-dependent activation of apoptotic pathway. Degradation of TIP60, therefore, allows low- and high-risk HPV to promote cell proliferation and cell survival.

Degradation of Tyrosine Phosphatase PTPN3 (PTPH1) by Association with Oncogenic Human Papillomavirus E6 Proteins

ABSTRACT Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation.Human papillomavirus (HPV) E6 oncoproteins have been previously shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). Here we show that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains previously implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase.

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins.

Targeting HPV Papillomaviruses infect mammalian epithelial cells and induce cancers, including cervical cancer in humans. Vaccines against human papillomavirus (HPV) can prevent, but not cure, infection. A key viral oncoprotein, E6, acts by binding and inactivating many host proteins. Zanier et al. (p. 694) determined high-resolution crystal structures of bovine papillomavirus bound to a peptide from the focal adhesion protein, paxillin, and of HPV bound to a peptide from the ubiquitin ligase E6AP. The structures show that the peptide binds in a pocket formed by two zinc domains and a linker helix, which represents a promising target for therapeutics. Crystal structures show how a key oncoprotein in human papillomavirus binds host proteins. E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins.

Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

The p53 pro-apoptotic tumour suppressor is mutated or functionally altered in most cancers. In epithelial tumours induced by ‘high-risk’ mucosal human papilloma viruses, including human cervical carcinoma and a growing number of head-and-neck cancers, p53 is degraded by the viral oncoprotein E6 (ref. 2). In this process, E6 binds to a short leucine (L)-rich LxxLL consensus sequence within the cellular ubiquitin ligase E6AP. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 (ref. 4). Neither E6 nor E6AP are separately able to recruit p53 (refs 3, 5), and the precise mode of assembly of E6, E6AP and p53 is unknown. Here we solve the crystal structure of a ternary complex comprising full-length human papilloma virus type 16 (HPV-16) E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6–p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumour suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against oncogenesis mediated by human papilloma virus.

The Stardust Family Protein MPP7 Forms a Tripartite Complex with LIN7 and DLG1 That Regulates the Stability and Localization of DLG1 to Cell Junctions

MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

Cooperative Transformation and Coexpression of Bovine Papillomavirus Type 1 E5 and E7 Proteins

ABSTRACT Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.

Identification of a Second Transforming Function in Bovine Papillomavirus Type 1 E6 and the Role of E6 Interactions with Paxillin, E6BP, and E6AP

ABSTRACT Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.

Peptide interactions stabilize and restructure human papillomavirus type 16 E6 to interact with p53.

ABSTRACT Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.

E6 proteins from diverse papillomaviruses self-associate both in vitro and in vivo.

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.