Structural Determinants of Ceruloplasmin Inflammatory Activity

铜蓝蛋白炎症活性的结构决定因素

基本信息

批准号：
6853420
负责人：
PAUL L FOX
金额：
$ 38.19万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2009-03-31
项目状态：
已结题

项目摘要

Inflammation and macromolecule oxidation are inter-related processes that are major contributors to atherogenesis. An important role of Cp in inflammation is suggested by its increased plasma level during the acute phase reaction and by its synthesis by activated macrophages in sites of inflammation. Clinical studies have shown that elevated plasma Cp is a significant risk factor for atherosclerosis, restenosis after endarterectomy, and myocardial infarction. In view of our finding that Cp copper induces oxidative modification of LDL in vitro, Cp may represent an important molecular link connecting inflammation, oxidation, and atherosclerosis. In new preliminary studies, we show that Cp binds with high affinity to LDL, and that the interaction is required for LDL oxidation by Cp. Other preliminary evidence indicates that Cp binds LDL in human plasma, and that binding to LDL may be higher in patients with cardiovascular disease. Studies with epitope-specific antibodies to Cp localized the interaction site to the second major sequence domain of Cp. These structural studies may be relevant to human inflammatory diseases, e.g., atherosclerosis, since coding-altering, single nucleotide polymorphisms (SNPs) have been identified in the human Cp gene, and two are adjacent to the Cp/LDL binding site. Preliminary studies in mice with targeted deletion of the Cp gene suggests that Cp increases oxidative modification of proteins and lipids in inflammatory sites in vivo. From previous work and our new preliminary results we propose the following hypothesis: That in sites of inflammation, macrophage-derived Cp binds to LDL and causes specific copper ion-mediated oxidative modification of the protein and lipid components. We further propose that by defining the specific Cp domains and amino acids required for pro- and anti-inflammatory activities, recombinant, monofunctional Cp can be generated that can test the function of the individual activities in vivo. Finally, we propose that human Cp containing SNPs in these functional domains alter the inflammatory properties of Cp, and also the risk for atherosclerosis in humans. We will test this hypothesis by pursuing the following Specific Aims: Aim 1. Determine the structural requirements for pro- and anti-inflammatory Cp activities; we will analyze structural determinants of Cp required for LDL oxidase, ferroxidase, and nitrosation activities, and for Cp interaction with LDL. Aim 2. Investigate the role of Cp in oxidation during inflammatory processes in vivo. Cp-null mice will be subjected to sepsis and peritonitis models of inflammation, and specific lipid and protein oxidation products in peritoneal lavage and plasma will be measured by LC-mass spectrometry These studies will elucidate, at the molecular, animal, and patient level, the role of Cp activities in inflammation.

炎症和大分子氧化是相互关联的过程，是动脉粥样硬化形成的主要原因。 Cp 在炎症中的重要作用是通过其在急性期反应期间血浆水平的增加以及通过炎症部位活化的巨噬细胞的合成来表明的。临床研究表明，血浆Cp升高是动脉粥样硬化、动脉内膜切除术后再狭窄和心肌梗塞的重要危险因素。鉴于我们发现 Cp 铜在体外诱导 LDL 氧化修饰，Cp 可能代表了连接炎症、氧化和动脉粥样硬化的重要分子联系。在新的初步研究中，我们表明 Cp 以高亲和力与 LDL 结合，并且这种相互作用是 Cp 氧化 LDL 所必需的。其他初步证据表明 Cp 与 LDL 结合人类血浆中，心血管疾病患者与 LDL 的结合可能更高。对 Cp 表位特异性抗体的研究将相互作用位点定位于 Cp 的第二个主要序列域。这些结构研究可能与人类炎症性疾病（例如动脉粥样硬化）相关，因为已在人类 Cp 基因中鉴定出编码改变的单核苷酸多态性 (SNP)，并且其中两个与 Cp/LDL 结合位点相邻。对靶向删除 Cp 基因的小鼠进行的初步研究表明，Cp 会增加体内炎症部位蛋白质和脂质的氧化修饰。根据之前的工作和我们新的初步结果，我们提出以下假设：在炎症部位，巨噬细胞衍生的 Cp 与 LDL 结合并引起特定的铜离子介导的氧化蛋白质和脂质成分的修饰。我们进一步建议，通过定义促炎和抗炎活性所需的特定 Cp 结构域和氨基酸，可以生成重组的单功能 Cp，可以测试体内各个活性的功能。最后，我们提出，在这些功能域中含有 SNP 的人类 Cp 会改变 Cp 的炎症特性，以及人类动脉粥样硬化的风险。我们将通过追求以下具体目标来检验这一假设：目标 1. 确定促炎和抗炎 Cp 活性的结构要求；我们将分析结构 LDL 氧化酶、亚铁氧化酶和亚硝化活性以及 Cp 与 LDL 相互作用所需的 Cp 决定因素。目标 2. 研究 Cp 在体内炎症过程中氧化中的作用。 Cp-null 小鼠将受到脓毒症和腹膜炎炎症模型的影响，腹腔灌洗液和血浆中的特定脂质和蛋白质氧化产物将通过 LC 质谱法进行测量。这些研究将在分子、动物和患者水平上阐明， Cp 活性在炎症中的作用。