Atherogenic Effects of Tyrosine Oxidation in HDL

描述（由申请人提供）：HDL载脂蛋白（特别是apoA-I）与细胞膜转运蛋白ABCA 1的相互作用可清除过量的细胞胆固醇并防止动脉粥样硬化形成。这一过程是由从头合成或从HDL颗粒解离产生的贫脂载脂蛋白介导的。因此，损害apoA-I的生成或脂质流出活性的因素可能具有深刻的致动脉粥样硬化作用。氧化损伤与动脉粥样硬化（一种慢性炎症性疾病）的发病机制有关。3-在人类动脉粥样硬化病变中检测到由吞噬细胞衍生的次氯酸（HOCI）和活性氮物质（RNS）产生的蛋白质氧化的稳定产物氯酪氨酸和3-硝基酪氨酸。然而，控制蛋白质中酪氨酸氧化的潜在因素仍然知之甚少。我们发现HDL相关的和游离的apoA-I被HOCI和过氧亚硝酸盐氧化会损害其通过ABCA 1途径清除细胞胆固醇的能力，这与HDL载脂蛋白在体内氧化会致动脉粥样硬化的可能性一致。我们建议测试的假设，即位点特异性氧化酪氨酸残基的吞噬细胞衍生的HOCI和RNS改变HDL的生物和动脉粥样硬化保护功能。我们将表征apoA-I中酪氨酸的位点特异性氧化对其去除细胞胆固醇的能力的影响，鉴定通过HOCI和RNS指导位点特异性酪氨酸氧化的蛋白质基序，并确定HDL是否是动脉壁氧化修饰的生理靶点。该项目将使用HPLC和串联质谱法定位和定量apoA-I和模型肽中酪氨酸氧化的位点，使用细胞生物学方法表征apoA-I氧化对脂质转运活性的影响以及与ABCA 1 apo A-I诱变的相互作用，以测试位点特异性修饰的功能意义，使用组织分析来鉴定和表征人类动脉粥样硬化病变中氧化的apoA-I，和小鼠模型方法来测试巨噬细胞产生的HOCI和RNS对体内apoA-I氧化和动脉粥样硬化形成的作用。拟议的研究将提供深入了解的结构特征，直接氧化修饰的蛋白质，具有重要意义的氧化反应在动脉粥样硬化和其他炎症性疾病的生理意义。

DESCRIPTION (provided by applicant): The interaction of HDL apolipoproteins, particularly apoA-I, with the cell membrane transporter ABCA1 removes excess cellular cholesterol and protects against atherogenesis. This process is mediated by lipid-poor apolipoproteins generated by either de novo synthesis or dissociation from HDL particles. Thus, factors that impair the generation or lipid efflux activity of apoA-I could have profound atherogenic effects. Oxidative damage is implicated in the pathogenesis of atherosclerosis, a chronic inflammatory disease. 3-Chlorotyrosine and 3-nitrotyrosine, stable products of protein oxidation generated by phagocyte-derived hypochlorous acid (HOCI) and reactive nitrogen species (RNS), have been detected in human atherosclerotic lesions. However, the underlying factors that control tyrosine oxidation in proteins remain poorly understood. We found that oxidation of HDL-associated and free apoA-I by HOCI and peroxynitite impairs its ability to remove cellular cholesterol by the ABCA1 pathway, consistent with the possibility that oxidation of HDL apolipoproteins in vivo would be atherogenic. We propose to test the hypothesis that site-specific oxidation of tyrosine residues by phagocyte-derived HOCI and RNS alters the biological and atheroprotective function of HDL. We will characterize the effects of site-specific oxidation of tyrosines in apoA-I on its ability to remove cellular cholesterol, identify protein motifs that direct site-specific tyrosine oxidation by HOCI and RNS, and determine if HDL is a physiological target for oxidative modification in the artery wall. This project will use HPLC and tandem mass spectrometry to locate and quantify the sites of tyrosine oxidation in apoA-I and model peptides, cell biology procedures to characterize the effects of apoA-I oxidation on lipid transport activity and interactions with ABCA1 apo A-I mutagenesis to test the functional significance of site-specific modifications, tissue analysis to identify and characterize oxidized apoA-I in human atherosclerotic lesions, and mouse model approaches to test for the effects of macrophage-generated HOCI and RNS on apoA-I oxidation and atherogenesis in vivo. The proposed studies will provide insights into the structural features that direct oxidative modification of proteins, with important implications for the physiological significance of oxidative reactions in atherosclerosis and other inflammatory diseases.