Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers

Abstract : We have previously identified a peptide aptamer (named "R5G42") via functional selection, for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait", allowed identification of two binding proteins with very different functions: Calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized e.g. for its role in the immune response, and NS5A-TP2/HDDC2, a much less studied protein, induced subsequent to Hepatitis C virus Non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective, in the present study, was to dissect the dual target specificity of R5G42 in order to have tools with which to be able to better characterize the actions of the peptide aptamers towards their individual targets. This was achieved by the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity towards CnA and NS5A-TP2, and analyzing their sequence. An interdisciplinary approach, involving biomolecular computer simulations with integration of the sequence data and yeast two hybrid binding phenotypes of these mutants, yielded two structurally-distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- vs NS5A-TP2-specific peptide aptamers indicated that, while both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate nuclear translocation of NFAT, indicative of activation of endogenous CnA. By dissecting target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independently of binding to NS5A-TP2 and will be an important tool to study the role of CnA activation in the regulation of different signalling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.
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Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2013, 12 (7), pp.1939-52. 〈10.1074〉
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Contributeur : Agnès Ganivet <>
Soumis le : mercredi 19 mars 2014 - 10:12:30
Dernière modification le : jeudi 8 février 2018 - 11:10:16

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Silvia Dibenedetto, David Cluet, Pierre-Nicolas Stebe, Véronique Baumlé, Jérémie Léault, et al.. Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers. Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2013, 12 (7), pp.1939-52. 〈10.1074〉. 〈ensl-00960932〉

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