Dataset Information

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex.

ABSTRACT: In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ∼13 μs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47R^E6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47^E6 and R290^p53, which is unfavorable for E6-p53 binding. R102A^E6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47^E6 and E286^p53/L298^p53/R290^p53. L50E^E6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.

SUBMITTER: Li L

PROVIDER: S-EPMC9117921 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex.

Li Le L Dong Xuewei X Tang Yiming Y Lao Zenghui Z Li Xuhua X Lei Jiangtao J Wei Guanghong G

Biophysical journal 20220329 9

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Here ...[more]

PMID: 35364103

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Project description:The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Project description:BackgroundOur 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 findingsThe 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/significanceThe 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.

Dataset Information

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex.

Publications

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex.

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