Endothelial activation by an isoprostane phospholipid

异前列烷磷脂激活内皮细胞

基本信息

批准号：
7626445
负责人：
Judith Anne Berliner
金额：
$ 30.8万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7626445
关键词：
Amines Amino Acid Sequence Amino Acids Arterial Fatty Streak Atherosclerosis Binding Binding Sites Blood Vessels Cell Differentiation process Cell physiology Chemistry Chronic Coagulants Collaborations Dendritic Cells Disease Drug Delivery Systems Electrospray Ionization Endothelial Cells Epoxy Compounds Genes Genetic Transcription Goals Grant Guanidines HRAS gene Inflammation Inflammatory Inflammatory Response Integrins Isomerism Isoprostanes Knowledge Lead Learning Lecithin Lipids Mediating Membrane Membrane Proteins Messenger RNA Methods Mining Modeling Nonesterified Fatty Acids Oxidative Stress Pathway interactions Peptides Permeability Phospholipids Preparation Protein Binding Proteins Reaction Recombinant Proteins Site Structure Sulfhydryl Compounds Tern Testing Upper arm Vascular Endothelial Growth Factor Receptor-2 adduct analog base design functional group high throughput screening human RIPK1 protein insight macrophage monocyte oxidized phosphatidyl choline public health relevance receptor response scale up tandem mass spectrometry

项目摘要

DESCRIPTION (provided by applicant): 1-Palmitoyl-2-(5,6-epoxyisoprostanoyl E )-sn-glycero-3-phosphatidylcholine (PEIPC) is emerging as a major regulator of vascular cell function. In endothelial cells it has been demonstrated to increase inflammation, procoagulant responses, to regulate junction permeability and to increase oxidative stress. In macrophages it has been demonstrated to regulate dendritic cell differentiation. The goal of the proposed studies is to synthesize sufficient quantities of the most active diastereomer in order to gain insight into the mechanism of action of PEIPC. In previous studies we synthesized one isomer of PEIPC consistent with the NMR of the natural PEIPC. In Aim 1 we will now synthesize the diasteromer and identify the most active of the two isomers in regulating endothelial cell responses. We have observed that PEIPC can covalently bind to at least 20 endothelial cell proteins. In Aim 2 we will identify the PEIPC functionality (likely the enone or the epoxide) that interacts with proteins. We will begin by examining the interaction with functional groups of amino acids, then examine the interaction of the most active functional group of PEIPC with peptides. Using two model proteins that bind PEIPC, VEGFR2 and H-ras, we will determine the amino acid sequence involved in binding of PEIPC or its free fatty acid. Using electrospray ionization-tandem mass spectrometry, unique fragmentation spectra produced by these specific lipid-protein interactions will be identified for use in discovering additional endothelial cell protein targets of PEIPC. Armed with this knowledge of the mechanism of the covalent binding of PEIPC to model proteins, we will synthesize analogues of PEIPC and test their effects on PEIPC action. We have previously determined that activation of VEGFR2 is required for the OxPAPC and PEIPC mediated activation of ERK and SREBP. We have also determined that inactivation of H-Ras is necessary for the activation of beta one integrins that lead to monocyte binding. Effects of antagonists on these responses will be determined. Overall these studies will define the chemistry of the PEIPC interaction with selected proteins that control endothelial cell function and will test the hypothesis that covalent binding of PEIPC activates pathways that control the endothelial cell inflammatory response. PUBLIC HEALTH RELEVANCE: Oxidized phospholipids, which accumulate in atherosclerotic lesions and other chronic inflammatory sites, have been shown to be important regulators of endothelial cell inflammatory and pro-coagulant responses that contribute to atherosclerosis. These studies will gain insight into how a particular oxidized phospholipid, PEIPC, causes this activation and will develop analogues to inhibit activation. These studies thus may provide a new drug target for atherosclerosis and other chronic inflammatory diseases.

描述（由申请人提供）：1-棕榈酰-2-(5,6-环氧异前列腺酰E)-sn-甘油-3-磷脂酰胆碱(PEIPC)正在成为血管细胞功能的主要调节剂。在内皮细胞中，它已被证明会增加炎症、促凝血反应、调节连接通透性并增加氧化应激。在巨噬细胞中，它已被证明可以调节树突细胞分化。拟议研究的目标是合成足够数量的最活跃的非对映异构体，以深入了解 PEIPC 的作用机制。在之前的研究中，我们合成了一种与天然 PEIPC 的 NMR 一致的 PEIPC 异构体。在目标 1 中，我们现在将合成非对映异构体并鉴定两种异构体中在调节内皮细胞反应中最活跃的异构体。我们观察到 PEIPC 可以与至少 20 种内皮细胞蛋白共价结合。在目标 2 中，我们将识别与蛋白质相互作用的 PEIPC 功能（可能是烯酮或环氧化物）。我们将首先检查与氨基酸官能团的相互作用，然后检查 PEIPC 最活跃的官能团与肽的相互作用。使用结合 PEIPC、VEGFR2 和 H-ras 的两种模型蛋白，我们将确定参与 PEIPC 或其游离脂肪酸结合的氨基酸序列。使用电喷雾电离串联质谱法，将鉴定由这些特定脂质-蛋白质相互作用产生的独特碎片光谱，用于发现 PEIPC 的其他内皮细胞蛋白质靶点。有了 PEIPC 与模型蛋白共价结合机制的知识，我们将合成 PEIPC 类似物并测试它们对 PEIPC 作用的影响。我们之前已经确定 VEGFR2 的激活是 OxPAPC 和 PEIPC 介导的 ERK 和 SREBP 激活所必需的。我们还确定，H-Ras 的失活对于激活导致单核细胞结合的 β1 整联蛋白是必要的。将确定拮抗剂对这些反应的影响。总的来说，这些研究将定义 PEIPC 与控制内皮细胞功能的选定蛋白质相互作用的化学性质，并将检验 PEIPC 的共价结合激活控制内皮细胞炎症反应的途径的假设。公共健康相关性：氧化磷脂在动脉粥样硬化病变和其他慢性炎症部位积聚，已被证明是导致动脉粥样硬化的内皮细胞炎症和促凝血反应的重要调节剂。这些研究将深入了解特定的氧化磷脂 (PEIPC) 如何引起这种激活，并将开发类似物来抑制激活。因此，这些研究可能为动脉粥样硬化和其他慢性炎症疾病提供新的药物靶点。