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 Apta 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 their specificity 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 the first exogenous molecule reported, capable of directly activating CnA independently of binding to NS5A-TP2, whereas Apta-E1 is the first molecule reported that will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signalling pathways.

Calcineurin A versus NS5A-TP2/HD Domain Containing 2: A Case Study of Site-directed Low-frequency Random Mutagenesis for Dissecting Target Specificity of Peptide Aptamers / S., Dibenedetto; D., Cluet; P. N., Stebe; V., Baumle; J., Leault; R., Terreux; M., Bickle; B. D. E., Chassey; I., Mikaelian; P., Colas; M., Spichty; Zoli, Michele; B. B., Rudkin. - In: MOLECULAR & CELLULAR PROTEOMICS. - ISSN 1535-9476. - STAMPA. - 12:7(2013), pp. 1939-1952. [10.1074/mcp.M112.024612]

Calcineurin A versus NS5A-TP2/HD Domain Containing 2: A Case Study of Site-directed Low-frequency Random Mutagenesis for Dissecting Target Specificity of Peptide Aptamers

ZOLI, Michele;
2013

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 Apta 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 their specificity 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 the first exogenous molecule reported, capable of directly activating CnA independently of binding to NS5A-TP2, whereas Apta-E1 is the first molecule reported that will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signalling pathways.
2013
12
7
1939
1952
Calcineurin A versus NS5A-TP2/HD Domain Containing 2: A Case Study of Site-directed Low-frequency Random Mutagenesis for Dissecting Target Specificity of Peptide Aptamers / S., Dibenedetto; D., Cluet; P. N., Stebe; V., Baumle; J., Leault; R., Terreux; M., Bickle; B. D. E., Chassey; I., Mikaelian; P., Colas; M., Spichty; Zoli, Michele; B. B., Rudkin. - In: MOLECULAR & CELLULAR PROTEOMICS. - ISSN 1535-9476. - STAMPA. - 12:7(2013), pp. 1939-1952. [10.1074/mcp.M112.024612]
S., Dibenedetto; D., Cluet; P. N., Stebe; V., Baumle; J., Leault; R., Terreux; M., Bickle; B. D. E., Chassey; I., Mikaelian; P., Colas; M., Spichty; Z...espandi
File in questo prodotto:
File Dimensione Formato  
1-s2.0-S1535947620325615-main.pdf

Open access

Tipologia: Versione pubblicata dall'editore
Dimensione 2.03 MB
Formato Adobe PDF
2.03 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/973495
Citazioni
  • ???jsp.display-item.citation.pmc??? 0
  • Scopus 1
  • ???jsp.display-item.citation.isi??? 0
social impact