Lipid Modification of Proteins by the PGH Synthases

拟议研究的广泛目标是扩大我们对 环氧合酶(Coxs、前列腺素H合成酶)。这些酶重要地参与了 许多生理功能和疾病，包括导致血栓形成的关键参与 心肌梗死和中风，在致癌方面，防止胃肠道溃疡，以及 发炎。这项研究解决了蛋白质氧化所产生的蛋白质的脂质修饰问题。 花生四烯酸由前列腺素H(PGH)合成酶合成。PGH合成酶的产物PGH2可以 部分重排生成左旋糖苷(LG)-酮醛，它是活性最强的化合物之一 生物分子。LG与蛋白质的赖氨酸残基反应产生蛋白质的共价加合物。 本课题组论证了LG与赖氨酸反应生成稳定的LG-赖氨酸内酰胺加合物结构 允许用LC/串联质谱仪分析与蛋白质加成的LG。我们有 上皮细胞中血小板PGHS-1和PGHS-2上LG加合物的形成 其他蛋白质。我们已经开发出清道夫，可以阻断细胞中蛋白质LG加合物的形成。 而不会抑制PGHS。利用这些食腐动物作为工具，我们的初步研究形成了 假设在PGHS上形成LG加合物有助于已知的加速 花生四烯酸对这些酶的降解。为了验证这一假说，建议进行调查。 并确定PGHS上LG引用的位置。红曲霉菌中蛋白水解肽的鉴定 采用串联质谱学的方法，利用两种方法进行加合后的光谱分析。 为了丰富和浓缩这些脂质修饰的多肽；针对LG-赖氨酸内酰胺残基的抗体将是 用于捕获加成的多肽，这种方法将由一种新的方法来补充 以β-烯基-花生四烯酸为底物分离加合物和捕获加合物 多肽与点击化学，然后光裂解释放多肽。这项研究将 用左旋糖胺产物和意志表征PGHS的翻译后修饰位点 确定这些修饰对这些重要酶的降解的影响。

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.