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Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.

We have identified a virus-activated factor (VAF) that binds to a regulatory element shared by different virus-inducible genes. We provide evidence that VAF contains two members of the interferon regulatory factor (IRF) family of transcriptional activator proteins (IRF-3 and IRF-7), as well as the transcriptional coactivator proteins p300 and CBP. Remarkably, VAF, as well as recombinant IRF-3 and IRF-7 proteins, binds very weakly to the interferon-beta (IFN-beta) gene promoter in vitro. However, in virus-infected cells, both proteins are recruited to the endogenous IFN-beta promoter as part of a protein complex that includes ATF-2/c-Jun and NF-kappa B. These observations provide a unique example of the coordinate activation of multiple transcriptional activator proteins and their highly cooperative assembly into a transcriptional enhancer complex in vivo.

The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitinmediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an -350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).The hallmark of the ubiquitin-mediated proteolytic pathway is the covalent attachment of the 76-aa ubiquitin polypeptide to target proteins, through isopeptide bond formation between the carboxyl terminus of ubiquitin and the s-amino group of one or more lysine residues on the protein substrate (reviewed in refs. 1 and 2). Additional ubiquitin moieties can be ligated via lysine residues of ubiquitin itself, resulting in the formation of multiubiquitinated proteins, which are then recognized and degraded by the 26S protease complex. While much of the biochemistry of the ubiquitin proteolysis system has been elucidated, a basic question has remained largely unanswered: how are proteins specifically recognized and targeted for ubiquitination?Protein ubiquitination involves three classes of enzymes. Ubiquitin is activated by the El ubiquitin-activating enzyme in an ATP-dependent reaction, resulting in thioester formation between a specific cysteine of the enzyme and the carboxyl terminus of ubiquitin. The activated ubiquitin is transferred to a cysteine residue of one of a number of low molecular weight E2 ubiquitin-conjugating enzymes. The E2 proteins have generally been thought to catalyze the final ubiquitination of the substrate protein, often in conjunction with a third group of proteins, the E3 ubiquitin-protein ligases. E3 activities have been proposed to play a major role in defining the substrate specificity of the ubiqulitin system, perhaps through direct binding of substrates. In the yeast Saccharomyces cerevisiae at least 12 different E2 genes have been identified (1). Only two known E3 genes have been cloned, yeast UBR1 (3) and the human E6-AP gene (4, 5).E6-AP was discovered in the course of c...

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Bromodomain protein 4 (Brd4) plays critical roles in development, cancer progression, and virus-host pathogenesis. To gain mechanistic insight into the various biological functions of Brd4, we performed a proteomic analysis to identify and characterize Brd4-associated cellular proteins. We found that the extraterminal (ET) domain, whose function has to date not been determined, interacts with NSD3, JMJD6, CHD4, GLTSCR1, and ATAD5. These ET-domain interactions were also conserved for Brd2 and Brd3, the other human BET proteins tested. We demonstrated that GLTSCR1, NSD3, and JMJD6 impart a pTEFb-independent transcriptional activation function on Brd4. NSD3 as well as JMJD6 is recruited to regulated genes in a Brd4-dependent manner. Moreover, we found that depletion of Brd4 or NSD3 reduces H3K36 methylation, demonstrating that the Brd4/NSD3 complex regulates this specific histone modification. Our results indicate that the Brd4 ET domain through the recruitment of the specific effectors regulates transcriptional activity. In particular, we show that one of these effectors, NSD3, regulates transcription by modifying the chromatin microenvironment at Brd4 target genes. Our study thus identifies the ET domain as a second important transcriptional regulatory domain for Brd4 in addition to the carboxyl-terminal domain (CTD) that interacts with pTEFb.One mechanism underlying the regulation of gene expression is the targeting of multiprotein complexes to modified histones, which then alters the chromatin microenvironment to stimulate or inhibit gene expression. The bromodomains and extraterminal (BET) domain family of proteins are characterized by the presence of two conserved domains, the tandem, amino-terminal bromodomains (BDI and BDII), which bind acetylated chromatin, and an extraterminal (ET) domain, whose function is unknown. The BET family is conserved from yeast to mammals and includes Saccharomyces cerevisiae bromodomain factor 1 (bdf1) and bromodomain factor 2 (bdf2), Drosophila melanogaster female sterile homeotic [fs(1)h], and mammalian Brd2, Brd3, Brd4, and testes/oocyte-specific BrdT/ Brd6. In yeast, deletion of bdf1 leads to a reduced growth rate and deletion of both bdf1 and bdf2 is lethal (27). Mutations of fs(1)h cause segmental abnormalities, including missing organs and homeotic transformations in the progeny of mutant females in Drosophila (13). Knockout of Brd4 or Brd2 in mice results in early embryonic lethality (18,21).The BET proteins have been shown to be important players in human disease, including viral infections and cancer. Several different viruses target the individual BET proteins for a variety of purposes but often to regulate viral and cellular transcription (4,7,31,37,41,45,57,60). The papillomavirus E2 proteins bind to Brd4, and some utilize this interaction in tethering the viral genomes to mitotic chromosomes (1,3,57,59). The papillomavirus E2 transcriptional activation functions are also mediated through Brd4 (35,41,42). With regard to human cancer, the Brd4-NUT and Brd3-NUT fusio...