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Zanier K.,Institute Of Recherche Of Lecole Of Biotechnologie Of Strasbourg Irebs | Stutz C.,German Cancer Research Center | Kintscher S.,German Cancer Research Center | Reinz E.,German Cancer Research Center | And 5 more authors.
PLoS ONE | Year: 2014

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, 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/pep11interaction 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. © 2014 Zanier et al. Source

Zanier K.,Institute Of Recherche Of Lecole Of Biotechnologie Of Strasbourg Irebs | M'Hamed Ould Sidi A.O.,Institute Of Recherche Of Lecole Of Biotechnologie Of Strasbourg Irebs | Boulade-Ladame C.,Institute Of Recherche Of Lecole Of Biotechnologie Of Strasbourg Irebs | Rybin V.,European Molecular Biology Laboratories EMBL | And 4 more authors.
Structure | Year: 2012

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. © 2012 Elsevier Ltd. Source

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