We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529 -531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second ␣ helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. The transforming properties of HPVs reside in two genes, E6 and E7. The E6 and E7 genes are consistently expressed in HPV-positive cervical cancers and derived cell lines (5-7). They cooperate to immortalize primary human keratinocytes (8 -13). HPV-16 E6 also cooperates with activated Ras in the transformation and immortalization of baby mouse kidney cells and baby rat kidney cells (14, 15). Independently of E7 or ras, HPV-16 E6 can transform NIH 3T3 cells (16), immortalize human mammary epithelial cells (17), and induce keratinocyte resistance to calcium and serum-induced differentiation (18). The activity of E6 in different biological assays implies it may influence diverse cellular pathways.The ability of E6 protein to associate with the cellular tumor suppressor p53 has been suggested as the mechanism by which the viral protein promotes cell growth and proliferation (19). Although binding of high risk HPV E6s with p53 appears to be mediated by another cellular protein, E6AP (20), direct in vitro association of E6 with p53 has also been observed (21,22). The complex of E6 and E6AP functions as an ubiquitin-protein ligase that results in the specific ubiquitination and subsequent degradation of p53 (23). Accumulating evidence suggests that E6 has functions independent of inactivating p53 in cellular transformation (24 -36).We have recently identified a cDNA encoding a cellular protein that binds papillomavirus E6 (E6BP or ERC-55) (1). E6BP was identified as a calcium-binding protein of the endoplasmic reticulum (37). The localization of E6BP is consistent with the localization of E6 to nonnuclear membranes (38). In vitro binding experiments demonstrated that E6BP interacted specifically with E6 proteins from cancer-related HPV types and the bovine papillomavirus type 1 (BPV-1). The transforming activity of a set of previously characterized BPV-1 E6 mutants correlated well with their E6BP binding ability. These results suggest that the E6BP interaction plays an important role for BPV-1 E6-induced transformation. More recently, it was reported that BPV-1 E6 associated with paxillin (39) as well as the trans-Golgi network-specific clathrin adaptor complex AP-1 (40) and that E6 proteins from some high risk HPVs interacts wit...
E6AP is a cellular protein that binds cancer-related papillomaviral E6 proteins. The E6 binding domain, called E6ap, is located on an 18-amino acid segment of E6AP. The corresponding peptide was synthesized and its structure determined by nuclear magnetic resonance spectroscopy. The overall structure of the peptide is helical. A consensus E6-binding sequence among different E6 interacting proteins contains three conserved hydrophobic residues. In the structure of the E6AP peptide, the three conserved leucines (Leu 9, Leu 12, and Leu 13) form a hydrophobic patch on one face of the alpha-helix. Substitution of any of these leucines with alanine abolished binding to E6 protein, indicating that the entire hydrophobic patch is necessary. Mutation of a glutamate to proline, but not alanine, also disrupted the interaction between E6 and E6AP protein, suggesting that the E6-binding motif of the E6AP protein must be helical when bound to E6. Comparison of the E6ap structure and mutational results with those of another E6-binding protein (E6BP/ERC-55) indicates the existence of a general E6-binding motif.
Atherogenesis involves cellular immune responses and altered vascular smooth muscle cell (SMC) function. Cytokines such as interleukin (IL)-1 alpha and interferon-gamma (IFN-gamma) may contribute to this process by activating SMC. To determine whether the anti-atherogenic mediator, nitric oxide (.NO), can modulate cytokine-induced SMC activation, we investigated the effects of various .NO-generating compounds on the expression of intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). Induction of ICAM-1 expression by IL-1 alpha and VCAM-1 expression by IFN-gamma was attenuated by .NO donors but not by cGMP analogues. Nuclear run-on assays and transfection studies using various VCAM-1 promoter constructs linked to the chloramphenicol acetyl-transferase reporter gene showed that .NO repressed IFN-gamma-induced VCAM-1 gene transcription, in part, through inhibition of nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assay revealed that SMC possess basal constitutive NF-kappa B activity, which was augmented by treatment with IL-1 alpha. In contrast, IFN-gamma induced and activated interferon regulatory factor (IRF)-1 but had little effect on basal constitutive NF-kappa B activity. .NO donors had no inhibitory effect on IRF-1 activation but did inhibit basal and IL-1 alpha-stimulated NF-kappa B activation. These findings suggest that the induction of ICAM-1 and VCAM-1 expression requires NF-kappa B activation and that .NO attenuates IFN-gamma-induced VCAM-1 expression primarily by inhibiting basal constitutive NF-kappa B activity in SMC.
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