Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.
ABSTRACT: The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.
Project description:Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.
Project description:Cervical cancer is a leading cause of death due to cancer among women worldwide. Using transgenic mice to dissect the contributions of the human papillomavirus (HPV) 16 E6 and E7 oncogenes in cervical cancer, E7 was identified previously to be the dominant oncogene. Specifically, when treated with exogenous estrogen for 6 months, E7 transgenic mice developed cancer throughout the reproductive tract, but E6 transgenic mice did not. E6 contributed to carcinogenesis of the reproductive tract, as E6/E7 double transgenic mice treated for 6 months with estrogen developed larger cancers than E7 transgenic mice. In the current study, we investigated whether the E6 oncogene alone could cooperate with estrogen to induce cervical cancer after an extended estrogen treatment period of 9 months. We found that the E6 oncogene synergizes with estrogen to induce cervical cancer after 9 months, indicating that E6 has a weaker but detectable oncogenic potential in the reproductive tract compared with the E7 oncogene. Using transgenic mice that express mutant forms of HPV16 E6, we determined that the interactions of E6 with cellular alpha-helix and PDZ partners correlate with its ability to induce cervical carcinogenesis. In analyzing the tumors arising in E6 transgenic mice, we learned that E6 induces expression of the E2F-responsive genes, Mcm7 and cyclin E, in the absence of the E7 oncogene. E6 also prevented the expression of p16 in tumors of the reproductive tract through a mechanism mediated by the interaction of E6 with alpha-helix partners.
Project description:Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys-->Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.
Project description:E7 is an accessory protein that is not encoded by all papillomaviruses. The E7 amino terminus contains two regions of similarity to conserved regions 1 and 2 of the adenovirus E1A protein, which are also conserved in the simian vacuolating virus 40 large tumor antigen. The E7 carboxyl terminus consists of a zinc-binding motif, which is related to similar motifs in E6 proteins. E7 proteins play a central role in the human papillomavirus life cycle, reprogramming the cellular environment to be conducive to viral replication. E7 proteins encoded by the cancer-associated alpha human papillomaviruses have potent transforming activities, which together with E6, are necessary but not sufficient to render their host squamous epithelial cell tumorigenic. This article strives to provide a comprehensive summary of the published research studies on human papillomavirus E7 proteins.
Project description:The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.
Project description:BACKGROUD:Variations in HPV LCR/E6/E7 have been shown to be associated with the viral persistence and cervical cancer development. So far, there are few reports about the polymorphisms of the HPV-58 LCR/E6/E7 sequences in Southwest China. This study aims to characterize the gene polymorphisms of the HPV-58 LCR/E6/E7 sequences in women of Southwest China, and assess the effects of variations on the immune recognition of viral E6 and E7 antigens. METHODS:Twelve LCR/E6/E7 of the HPV-58 isolates were amplified and sequenced. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED v3.3. The selection pressure acting on the HPV-58 E6 and E7 coding regions was estimated by Bayes empirical Bayes analysis of PAML 4.8. Meanwhile, the MHC class-I and II binding peptides were predicted by the ProPred-I server and ProPred server. The transcription factor binding sites in the HPV-58 LCR were analyzed using the JASPAR database. RESULTS:Twenty nine SNPs (20 in the LCR, 3 in the E6, 6 in the E7) were identified at 27 nucleotide sites across the HPV-58 LCR/E6/E7. From the most variable to the least variable, the nucleotide variations were LCR?>?E7?>?E6. The combinations of all the SNPs resulted in 11 unique sequences, which were clustered into the A lineage (7 belong to A1, 2 belong to A2, and 2 belong to A3). An insertion (TGTCAGTTTCCT) was found between the nucleotide sites 7280 and 7281 in 2 variants, and a deletion (TTTAT) was found between 7429 and 7433 in 1 variant. The most common non-synonymous substitution V77A in the E7 was observed in the sequences encoding the ?-helix. 63G in the E7 was determined to be the only one positively selected site in the HPV-58 E6/E7 sequences. Six non-synonymous amino acid substitutions (including S71F and K93 N in the E6, and T20I, G41R, G63S/D, and V77A in the E7) were affecting multiple putative epitopes for both CD4+ and CD8+ T-cells. In the LCR, C7265G and C7266T were the most variable sites and were the potential binding sites for the transcription factor SOX10. CONCLUSION:These results provide an insight into the intrinsic geographical relatedness and biological differences of the HPV-58 variants, and contribute to further research on the HPV-58 epidemiology, carcinogenesis, and therapeutic vaccine development.
Project description:More than 150 types of Human papillomaviruses (HPVs) have been isolated from numerous cutaneous and/or mucosal lesions. Flat wart samples on the face from 36 immunocompetent patients were collected and screened for HPV. From one sample, we cloned a putative novel genotype. The novel type consisted of 7779 bp in length with a GC content of 47.1%, containing open reading frames for putative early proteins (E1, E2, E4, E6, and E7) and two late proteins (L1 and L2). Homology searches and phylogenetic analyses indicated that it belonged to Alphapapillomavirus (Alpha-PV) species 2 and most closely resembled HPV 3. The virus fulfilled the definition of a novel type, and was named HPV 160 by the Reference Center for Papillomaviruses. The putative E7 protein of HPV 160 as well as HPV 29, 77, and 78 contained the Leu-X-Cys-X-Glu pRB-binding motif but other Alpha-PV species 2 (HPV 3, 10, 28, 94, 117, and 125) did not have this conserved motif.
Project description:The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to the heavy metals Cd(II), Zn(II), and Pb(II). Expression of this heavy-metal efflux pump is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. Here we report the X-ray crystal structure of CadC at 1.9 angstroms resolution. The dimensions of the protein dimer are approximately 30 angstroms by 40 angstroms by 70 angstroms. Each monomer contains six alpha-helices and a three-stranded beta-sheet. Helices 4 and 5 form a classic helix-turn-helix motif that is the putative DNA binding region. The alpha1 helix of one monomer crosses the dimer to approach the alpha4 helix of the other monomer, consistent with the previous proposal that these two regulatory metal binding sites for the inducer cadmium or lead are each formed by Cys-7 and Cys-11 from the N terminus of one monomer and Cys-58 and Cys-60 of the other monomer. Two nonregulatory metal binding sites containing zinc are formed between the two antiparallel alpha6 helices at the dimerization interface. This is the first reported three-dimensional structure of a member of the ArsR/SmtB family with regulatory metal binding sites at the DNA binding domain and the first structure of a transcription repressor that responds to the heavy metals Cd(II) and Pb(II).
Project description:His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s(-1) in α subunits and ∼10-fold faster in β subunits, ∼175,000 s(-1). Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is ∼80-90 μm(-1) s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, ∼0.08 μm(-1) s(-1). A transient intermediate with k'(CO) ≈ 0.7 μm(-1) s(-1) is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.
Project description:Human papillomavirus (HPV) E6 and E7 oncoproteins are critical for development and maintenance of the malignant phenotype in HPV-induced cancers. These two viral oncoproteins interfere with a plethora of cellular pathways, including the regulation of cell cycle and the control of apoptosis, which are critical in maintaining normal cellular functions. E6 and E7 bind directly with certain components of the Ubiquitin Proteasome System (UPS), enabling them to manipulate a number of important cellular pathways. These activities are the means by which HPV establishes an environment supporting the normal viral life cycle, however in some instances they can also lead to the development of malignancy. In this review, we have discussed how E6 and E7 oncoproteins from alpha and beta HPV types interact with the components of the UPS, and how this interplay contributes to the development of cancer.