Citrullination of ApoA1 as a Novel Contributor to Inflammatory Atherogenesis

 描述（由申请方提供）：瓜氨酸是肽基精氨酸翻译后转化为肽基瓜氨酸，由肽基精氨酸脱亚胺酶（PAD）家族介导。由于多形核白细胞以及单核细胞/巨噬细胞谱系的细胞组成型表达酶PAD 2和PAD 4，瓜氨酸已被证明是一种炎症依赖性过程。炎症和动脉粥样硬化之间的联系是建立良好的，涉及许多机制，包括髓过氧化物酶诱导的保护性HDL蛋白的氧化修饰。然而，最近在动脉粥样硬化斑块中鉴定的另一种翻译后修饰是瓜氨酸蛋白。我们已经鉴定了几种瓜氨酸化斑块蛋白，主要包括HDL相关蛋白ApoA-I。ApoA-I促进去除过量的细胞胆固醇，从而阻止动脉粥样硬化形成。此外，ApoA-I已被证明具有直接抗炎作用，从而限制血管炎症。我们建议研究瓜氨酸作为炎症诱导动脉粥样硬化进展的新介质。这项工作代表了我们先前研究的科学进展，证明了瓜氨酸在动脉粥样硬化斑块中的作用。这项研究将为白色血细胞普遍携带的一组酶-肽基精氨酸脱亚胺酶（PAD）如何促进动脉粥样硬化斑块内病理性炎症的影响提供新的见解。本申请的主要目的是研究ApoA-I瓜氨酸作为动脉粥样硬化发生和进展的风险因素的作用。具体而言，我们建议：1.从体外瓜氨酸化ApoA-I以及从人血浆分离的ApoA-I和动脉粥样硬化斑块内确定ApoA-I瓜氨酸酶的位点; 2.确认和表征ApoA-I胆固醇流出能力的瓜氨酸依赖性消除，并鉴定介导这种消除的关键残基; 3.使用药理学和遗传学动物模型来确定瓜氨酸在动脉粥样硬化进展中的作用，以及4.定量血浆ApoA-I中的瓜氨酸，并与亚临床动脉粥样硬化以及动脉粥样硬化事件相关。这些研究的成功可以确定炎症驱动动脉粥样硬化的新机制，并有可能有助于开发心血管风险预测的诊断测试，以及确定治疗和/或预防冠状动脉疾病的潜在治疗靶点。

 DESCRIPTION (provided by applicant): Citrullination is the post-translational conversion of peptidyl-arginine to peptidyl-citrulline and is mediated by a family of enzymes known as peptidyl-arginine deiminases (PADs). Citrullination has been demonstrated to be an inflammation-dependent process due to the constitutive expression of the enzymes PAD2 and PAD4 by polymorphonuclear leukocytes as well as cells of the monocytes/macrophage lineage. The link between inflammation and atherosclerosis is well established with numerous mechanisms implicated including oxidative modification of protective HDL proteins induced by the enzyme myeloperoxidase. However, another post- translation modification recently identified within the atherosclerotic plaque is protein citrullination. We have identified several citrullinated plaque proteins including prominently, the HDL-associated protein ApoA-I . ApoA-I facilitates removal of excess cellular cholesterol thus impeding atherogenesis. Additionally, ApoA-I has been demonstrated to have direct anti-inflammatory effects thus limiting vascular inflammation. We propose to study citrullination as a novel mediator of inflammation induced atherosclerotic progression. This work represents a scientific progression of our previous studies demonstrating citrullination within the atherosclerotic plaque. The proposed studies will provide new insights into how a group of enzymes ubiquitously carried by white blood cells, peptidyl arginine-deiminases (PADs), contributes to the effect of pathologic inflammation within the atherosclerotic plaque. The main goal of this application is to investigate the role of ApoA-I citrullination as a risk factor for atherosclerotic development and progression. Specifically, we propose to: 1. Define the sites of ApoA-I citrullination from in-vitro citrullinated ApoA-I as wellas ApoA-I isolated from human plasma, and from within the atherosclerotic plaque; 2. Confirm and characterize citrullination-dependent abrogation of ApoA-I cholesterol efflux capacity and to identify residues critical for mediating this abrogation; 3. Use pharmacologic and genetic animal models to define the role of citrullination in atherosclerosis progression, and 4. Quantitate citrullination of plasma ApoA-I and correlate with subclinical atherosclerosis as well as atherosclerotic events. Success of these studies could identify novel mechanisms contributing to inflammation-driven atherosclerosis and have the potential to both contribute to the development of diagnostic tests for cardiovascular risk prediction, as well as identifying potentia therapeutic targets for the treatment and/or prevention of coronary artery disease.