Structural Basis for Hijacking of Cellular LxxLL Motifs by Papillomavirus E6 Oncoproteins

Zanier, Katia; Charbonnier, Sébastian; Sidi, Abdellahi Ould M'hamed Ould; McEwen, A.G.; Ferrario, Maria Giovanna; Poussin-Courmontagne, Pierre; Cura, Vincent; Brimer, Nicole; Babah, Khaled Ould; Ansari, Tina; Muller, Isabelle S.; Stote, Roland H.; Cavarelli, Jean; Pol, Scott Vande; Travé, Gilles

doi:10.1126/science.1229934

Cited by 181 publications

(295 citation statements)

References 53 publications

(56 reference statements)

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“…Sequence alignments show that a number of HPV16 E6 residues that are critical for E6AP peptide and p53 binding are conserved in HPV49 E6, but not in HPV38 E6. We found that Arg55 and Arg131 of HPV16 E6, which provide crucial contacts to acidic residues of the LxxLL peptide of E6AP (25), are conserved (at least as positively charged residues) in HPV49 E6, but not in HPV38 E6. Similarly, the key p53-binding residue Asp49 (26) is conserved in HPV49, whereas it becomes an Asn residue in HPV38 E6 ( Supplementary Fig.…”

Section: Discussionmentioning

confidence: 83%

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer

et al. 2016

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The beta genus of human papillomaviruses (ß-HPV) includes approximately 50 different viral types that are subdivided into five species (ß-1 through ß-5). Nonmelanoma cancers may involve some ß-1 and ß-2 HPV types, but the biology of most ß-HPV types and their possible connections to human disease are still little characterized. In this study, we studied the effects of ß-3 type HPV49 in a novel transgenic (Tg) mouse model, using a cytokeratin K14 promoter to drive expression of the E6 and E7 genes from this virus in the basal skin epidermis and the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7-Tg mice). Viral oncogene expression only marginally increased cellular proliferation in the epidermis of Tg animals, compared with wild-type littermates, and we observed no spontaneous tumor formation during their entire lifespan. However, we found that K14 HPV49 E6/E7-Tg mice were highly susceptible to upper digestive tract carcinogenesis upon initiation with 4-nitroquinoline 1-oxide (4NQO). This was a selective effect, as the same mice did not exhibit any skin lesions after chronic UV irradiation. Opposite results were observed in an analogous Tg model expressing the ß-2 HPV38 E6 and E7 oncogenes at the same anatomic sites. While these mice were highly susceptible to UV-induced skin carcinogenesis, as previously shown, they were little affected by 4NQO treatment. Overall, our findings highlight important differences in the biologic properties of certain ß-type HPV that affect their impact on carcinogenesis in an anatomic site-specific manner.

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Section: Discussionmentioning

confidence: 83%

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer

et al. 2016

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“…5 In the same study we also reported the crystal structure of the E6 oncoprotein from Bovine Papillomavirus 1 (BPV1) bound to the LxxLL sequence from paxillin. In both complexes the LxxLL motif adopts a helical conformation and binds a hydrophobic-basic pocket, which is contributed by residues from the E6N and E6C domains, and from the interdomain helical linker.…”

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confidence: 97%

HPV-mediated inactivation of tumor suppressor p53

Travé

Zanier

2016

Cell Cycle

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“…Quality control can also be performed by mass spectrometry (to confirm the expected size and oxidation state) or chromatographic methods (to confirm purity or heterogeneity) 29 . Sometimes tag cleavage is unnecessary or even undesirable (particularly for poorly soluble proteins 30,31 ), so in this protocol cleavage is optional. Regardless, in all constructs there is a TEV protease cleavage site (ENLYFQ/[G] 32 ) directly preceding the target gene to produce native protein after cleavage (see Figure 2 and Discussion).…”

Section: Introductionmentioning

confidence: 99%

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

Saez

Nozach

Blémont

et al. 2014

JoVE

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Escherichia coli ( E. coli ) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project ( www.venomics.eu ), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

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Structural Basis for Hijacking of Cellular LxxLL Motifs by Papillomavirus E6 Oncoproteins

Cited by 181 publications

References 53 publications

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer

HPV-mediated inactivation of tumor suppressor p53

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

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