Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA(1): focus on vimentin

11 páginas, 4 figuras, 2 tablas -- PAGS nros. 474-1484Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A1 (PGA1) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA1 has been used as an inhibitor of transcription factor NF-κB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA1 could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA1 have been identified. In previous work, we have observed that a biotinylated analog of PGA1 that retains the cyclopentenone moiety (PGA1-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA1-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA1 addition. These results may contribute to a better understanding of the mechanism of action of PGA1 and the potential of cyPG-based therapeutic strategiesThis work was supported by grants SAF2006-03489 (Ministerio de Educación y Ciencia) and 0179/1 (Fundación La Caixa) to D.P.-S. J.G. is the recipient of a pre-doctoral fellowship from the I3P Program (C.S.I.C., Fondo Social Europeo). B. G. is the recipient of a fellowship from the FPI program (Ministerio de Educación y Ciencia). The stay of J.G. at the laboratory of J.T. was supported by EMBO and FEBS short term fellowships. The technical assistance of M.J. Carrasco is gratefully acknowledgedPeer reviewe