Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.     

The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP,Indicating Druggability of E6

The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.     

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.     

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.     

Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.     

GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

Background

Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis.

Methodology/Principal Findings

The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model.

Conclusion/Significance

The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.     

Human papillomavirus E6 regulates the cytoskeleton dynamics of keratinocytes through targeted degradation of p53

The attachment and spreading of keratinocyte cells result from interactions between integrins and immobilized extracellular matrix molecules. Human papillomavirus type 16 (HPV-16) E6 augmented the kinetics of cell spreading, while E6 genes from HPV-11 or bovine papillomavirus type 1 did not. The ability of E6 to interact with the E6AP ubiquitin ligase and target p53 degradation was required to augment cell-spreading kinetics; dominant negative p53 alleles also enhanced the kinetics of cell spreading and the level of attachment of cells to hydrophobic surfaces. The targeted degradation of p53 by E6 may contribute to the invasive phenotype exhibited by cervical cells that contain high-risk HPV types.     

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.     

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.     

<Emphasis Type="Italic">Human papillomavirus</Emphasis> type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

Background

Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women.Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated.During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet.Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins.

Methods

To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1.Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model.

Results

The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci.After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique.

Conclusions

Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

     

Non-human primate papillomavirus E6-mediated p53 degradation reveals ancient evolutionary adaptation of carcinogenic phenotype to host niche

Non-human primates (NHPs) are infected with papillomaviruses (PVs) closely related to their human counterparts, but there are few studies on the carcinogenicity of NHP-PVs. Using an in vitro cell co-transfection assay, we systematically screened the biochemical activity of E6 proteins encoded by macaque PVs for their ability to bind and promote degradation of host p53 proteins. A host species barrier exists between HPV16 and MfPV3 with respect to E6-mediated p53 degradation that is reversed when p53 residue 129 is swapped between human and macaque hosts. Systematic investigation found that E6 proteins encoded by most macaque PV types in the high-risk species α12, but not other Alpha-PV clades or Beta-/Gamma-PV genera, can effectively promote monkey p53 degradation. Interestingly, two macaque PV types (MfPV10 and MmPV1) can simultaneously inhibit the expression of human and monkey p53 proteins, revealing complex cross-host interactions between PV oncogenes and host proteomes. Single point-mutant experiments revealed that E6 residue 47 directly interacts with p53 residue 129 for host-specific degradation. These findings suggest an ancient host niche adaptation toward a carcinogenic phenotype in high-risk primate PV ancestors. Following periods of primate host speciation, a loss-of-function mutation model could be responsible for the formation of a host species barrier to E6-mediated p53 degradation between HPVs and NHP-PVs. Our work lays a genetic and functional basis for PV carcinogenicity, which provides important insights into the origin and evolution of specific pathogens in host pathogenesis.     

Targetting of the N‐terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric scFvs blocks the E6‐mediated degradation of cellular p53

The E6 protein of cancer‐associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6‐mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N‐terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6‐mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N‐terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein. Copyright © 1999 John Wiley & Sons, Ltd.     

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.     

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-γ-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4⁺ or CD8⁺ T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.     

Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis

Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution. E6 appears as a chameleon protein combining a conserved structural scaffold with highly variable surfaces participating in generic or specialized HPV functions. We investigated surface residues involved in two specialized activities of high-risk genital HPV E6: p53 tumor suppressor degradation and nucleic acid binding. Screening of E6 surface mutants identified an in vivo p53 degradation-defective mutant that fails to recruit p53 to ubiquitin ligase E6AP and restores high p53 levels in cervical carcinoma cells by competing with endogeneous E6. We also mapped the nucleic acid binding surface of E6, the positive potential of which correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understanding and counteracting the complex pathways of HPV-mediated pathogenesis.     

Degradation of p53 by Human Alphapapillomavirus E6 Proteins Shows a Stronger Correlation with Phylogeny than Oncogenicity

Background

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.

Principal Findings

We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.

Conclusions

These data suggest that the alpha-HPV E6 proteins'' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.