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（COX，前列腺素H-环氧化酶）。这些酶参与重要的 许多生理功能和疾病，包括关键参与血栓形成，导致 心肌梗死和中风，在致癌作用，保护胃肠道溃疡，和 炎症这项研究解决了蛋白质的脂质修饰，蛋白质是由氧化产生的， 前列腺素H（PGH）脱氢酶对花生四烯酸的作用。PGH内切酶的产物PGH 2可以 部分重排产生-酮醛，名为左甘定（LG），它是最具反应性的 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。 我们小组证明LG与赖氨酸反应产生稳定的LG-赖氨酸内酰胺加合物结构 允许通过LC/串联质谱法分析LG加合到蛋白质上。我们有 证明了LG加合物在血小板PGHS-1和上皮细胞PGHS-2上以及 其他蛋白质。我们已经开发了清除剂，阻止形成LG加合物的蛋白质在细胞中 而不抑制PGHS。利用这些清道夫作为工具，我们的初步研究已经形成了 一个假设的基础，即PGHS上LG加合物的形成有助于已知的加速 这些酶被花生四烯酸降解。调查提出了测试这一假设 并确定PGHS上LG内收的位点。鉴定来自大肠杆菌的蛋白水解肽 加合的PGHS将利用两种方法用串联质谱法进行 为了富集和浓缩这些脂质修饰的肽， 用于捕获加合肽，这种方法将通过一种新的方法来补充， 使用戊炔基-花生四烯酸作为底物分离加合物肽，并捕获加合物肽， 通过点击化学反应，然后光裂解以释放肽。这项研究将 表征PGHS通过其左旋兰素产物的翻译后修饰位点，并将 确定这些修饰对这些重要酶降解的影响。

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.