Lipid Modification of Proteins by the PGH Synthases

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The broad goal of the proposed research is to extend our understanding of the functions of the cyclooxygenase enzymes (COXs, prostaglandin H synthases). These enzymes participate importantly in many physiologic functions and diseases, including key participation in formation of the thrombi that lead to myocardial infarction and stroke, in carcinogenesis, protection from gastrointestinal ulceration, and inflammation. The research addresses the lipid-modification of proteins produced by the oxygenation of arachidonic acid by the prostaglandin H (PGH) synthases. The product of the PGH synthases, PGH2, can rearrange in part to yield the -ketoaldehydes named levuglandins (LG), which are among the most reactive biological molecules. Reaction of LG with lysine residues of proteins yields covalent adducts of the proteins. Demonstration by our group that reaction of LG with lysine yields a stable LG-lysine lactam adduct structure has permitted analysis of LG adducted to proteins by LC/Tandem mass spectrometry. We have demonstrated formation of LG adducts on platelet PGHS-1 and on PGHS-2 in epithelial cells as well as on other proteins. We have developed scavengers that block formation of LG adducts of proteins in cells without inhibiting the PGHSs. Utilizing these scavengers as tools, our preliminary studies have formed the basis for a hypothesis that formation of LG adducts on the PGHSs contributes to the known acceleration of the degradation of these enzymes by arachidonic acid. Investigations are proposed to test this hypothesis and to identify the sites on the PGHSs that are LG adducted. Identification of the proteolytic peptides from the PGHSs that are adducted wiil be carried out with tandem mass spectrometry, utilizing two approaches to enrich and concentrate these lipid-modified peptides; an antibody to LG-lysine lactam residues will be used to capture the adducted peptides, and this approach will be complemented by a novel method for isolating the adducted peptides using ¿-alkynl-arachidonic acid as substrate and capture of the adducted peptides with click chemistry followed by photocleavage to release the peptide. This research will characterize the sites of post-translational modification of the PGHSs by their levuglandin products and will determine the effect of these modifications on the degradation of these important enzymes.