title: Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA1: focus on vimentin.
abstract: Prostaglandins 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 A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-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 PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-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 PGA(1)-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 PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.
authors.affiliation: Cancer Proteomics Group, Ludwig Institute for Cancer Research, London, and Department of Gynaecological Oncology, University College London, UK.
authors.forename: Severine
authors.initials: S
authors.lastname: Gharbi
authors.forename: Beatriz
authors.initials: B
authors.lastname: Garzón
authors.forename: Javier
authors.initials: J
authors.lastname: Gayarre
authors.forename: John
authors.initials: J
authors.lastname: Timms
authors.forename: Dolores
authors.initials: D
authors.lastname: Pérez-Sala
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001360:Avidin / Q000737:chemistry; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000378:metabolism; D011454:Prostaglandins A / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D014746:Vimentin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000970:Antineoplastic Agents; D005904:Glial Fibrillary Acidic Protein; D018841:HSP90 Heat-Shock Proteins; D020648:Peptide Elongation Factor 1; D011454:Prostaglandins A; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D014746:Vimentin; C093483:neutravidin; D001360:Avidin; D049452:Green Fluorescent Proteins; D039601:Eukaryotic Initiation Factor-4A; D003545:Cysteine; C100573:prostaglandin A1