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This paper reports an approach to measure equilibrium binding affinities for interacting proteins in high throughput, allowing the rapid and quantitative profiling of the specificity of interaction motifs. Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

WebStructural details of how oncogenic human papilloma viruses induce cancer by targeting the tumour suppressor p53 for ubiquitin-mediated degradation. Viral hijack of p53 tumour suppressor Oncogenic human papillomaviruses induce cancer by targeting the tumour suppressor p53 for ubiquitin-mediated degradation. Katia Zanier and colleagues now reveal structural details of how this viral hijacking occurs. They solve the structure of a ternary complex revealing the interaction between the HPV16 oncoprotein E6, the LxxLL motif of the cellular ubiquitin ligase E6AP, and the core domain 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 1 , 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 3 . 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.

Cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) detects intracellular DNA and signals through the adapter protein STING to initiate the antiviral response to DNA viruses. Whether DNA viruses can prevent activation of the cGAS-STING pathway remains largely unknown. Here, we identify the oncogenes of the DNA tumor viruses, including E7 from human papillomavirus (HPV) and E1A from adenovirus, as potent and specific inhibitors of the cGAS-STING pathway. We show that the LXCXE motif of these oncoproteins, which is essential for blockade of the retinoblastoma tumor suppressor, is also important for antagonizing DNA sensing. E1A and E7 bind to STING, and silencing of these oncogenes in human tumor cells restores the cGAS-STING pathway. Our findings reveal a host-virus conflict that may have shaped the evolution of viral oncogenes.

Cervical cancer ranks as the third most prevalent malignancy in women worldwide. The persistence of Human papillomavirus (HPV) infection stands out as the foremost risk factor for cervical cancer development. Among the numerous HPV subtypes, HPV16 infection emerges as the primary pathogenic determinant of cervical cancer. To date, no specific drugs have been approved. In this study, we engineered two high-affinity fusion protein targeting HPV16 L1 protein based on the alpaca-derived single-domain antibody 2C12 previously obtained in our laboratory. These two fusion proteins exhibited potent neutralizing activity against HPV16 pseudovirus with IC50 values of 7.8 nM and 6.5 nM, respectively. Molecular docking analysis revealed that 2C12 formed ten pairs of hydrogen bonds with HPV16 L1, among which Arg39 and Thr100 established multiple pairs of hydrogen bonds with HPV16 L1, indicating their crucial roles in antigen-antibody binding process. These structural and biological findings underscore the effective binding capacity of these fusion proteins to HPV16, leading to reduced viral load and providing valuable insights into therapeutic antibody and vaccine development against HPV 16 infection.

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.

Human Papilloma Virus type 16 (HPV-16) is highly oncogenic with the E6 and E7 oncogenes playing crucial roles in the pathogenesis of HPV-related cervical carcinogenesis. Targeting these oncoproteins with specific inhibitors offers a promising approach for therapeutic intervention. This study aimed to identify potential inhibitors of the HPV-16 E6 and E7 oncoproteins through an in silico approach, providing a foundation for the development of targeted therapies against HPV associated malignancies. We performed virtual screening on a library of 1000 compounds to identify promising candidates. Subsequent molecular docking studies were conducted to assess the binding affinities of the promising candidates. The top-scoring compounds for oncoproteins were then subjected to molecular dynamics simulations to evaluate their stability and interaction profiles. The virtual screening identified 14 promising candidates followed by docking studies. Among these Galangin was identified as a promising inhibitor for the E6 oncogene, while Neoechinulin showed potential as an inhibitor of the E7 oncogene. Our findings suggest Galangin and Neoechinulin with high potential as therapeutic inhibitors of HPV-16 E6 and E7 oncogenes respectively. These inhibitors could contribute significantly to the development of targeted therapies against HPV associated malignancies. However, further in vitro and in vivo investigations are required to use these phytochemicals as antiviral agents against HPV-16.

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP. The human papillomavirus (HPV) E6 oncoprotein hijacks the ligase activity of the host E6AP to ubiquitinate the tumor suppressor p53. Here, the authors show how the presence of the HPV E6 oncoprotein transforms the inactive E6AP monomer into an active dimer, providing a structural understanding of the physiological and pathophysiological mechanisms of E6AP function.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

Human papillomavirus (HPV) induced cervical cancer is the second most common cause of death, after breast cancer, in females. Three prophylactic vaccines by Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK) have been confirmed to prevent high-risk HPV strains but these vaccines have been shown to be effective only in girls who have not been exposed to HPV previously. The constitutively expressed HPV oncoproteins E6 and E7 are usually used as target antigens for HPV therapeutic vaccines. These early (E) proteins are involved, for example, in maintaining the malignant phenotype of the cells. In this study, we predicted antigenic peptides of HPV types 16 and 18, encoded by E6 and E7 genes, using an immunoinformatics approach. To further evaluate the immunogenic potential of the predicted peptides, we studied their ability to bind to class I major histocompatibility complex (MHC-I) molecules in a computational docking study that was supported by molecular dynamics (MD) simulations and estimation of the free energies of binding of the peptides at the MHC-I binding cleft. Some of the predicted peptides exhibited comparable binding free energies and/or pattern of binding to experimentally verified MHC-I-binding epitopes that we used as references in MD simulations. Such peptides with good predicted affinity may serve as candidate epitopes for the development of therapeutic HPV peptide vaccines.

Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.