Structural Determinants of Ceruloplasmin Inflammatory Activity

铜蓝蛋白炎症活性的结构决定因素

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6853420
• 关键词: SDS polyacrylamide gel electrophoresis; atherosclerosis; binding sites; copper; enzyme activity; ferroxidase; gene expression; genetically modified animals; inflammation; laboratory mouse; liquid chromatography mass spectrometry; low density lipoprotein; macromolecule; oxidation; peritonitis; plasma; polymerase chain reaction; recombinant proteins; septicemia; single nucleotide polymorphism

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，是动脉粥样硬化的主要因素。 CP在炎症中的重要作用是在急性相反应期间的血浆水平升高以及炎症部位激活的巨噬细胞的合成。临床研究表明，血浆CP升高是动脉粥样硬化，内部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的氧化修饰，CP可能代表连接炎症，氧化和动脉粥样硬化的重要分子链接。在新的初步研究中，我们表明CP与LDL具有高亲和力结合，并且CP通过LDL氧化所必需的相互作用。其他初步证据表明CP结合了LDL 心血管疾病患者的人血浆以及与LDL的结合可能更高。 CP表位特异性抗体的研究将相互作用位点定位于CP的第二个主要序列结构域。这些结构研究可能与人类炎症性疾病有关，例如，动脉粥样硬化，因为在人CP基因中已经鉴定出了改变编码，单核苷酸多态性（SNP），而两种与CP/LDL结合位点相邻。对CP基因靶向缺失的小鼠的初步研究表明，CP会增加体内炎症部位的蛋白质和脂质的氧化修饰。从以前的工作和我们的新初步结果中，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氧化。 修饰蛋白质和脂质成分。我们进一步提出，通过定义促和抗炎活性所需的特定CP结构域和氨基酸，可以生成重组，单功能的CP，以测试体内各个活动的功能。最后，我们提出，这些功能结构域中含有SNP的人CP改变了CP的炎症特性，也改变了人类动脉粥样硬化的风险。我们将通过追求以下特定目标来检验这一假设：目标1。确定促和抗炎CP活动的结构要求；我们将分析结构 LDL氧化酶，铁氧化酶和亚硝化活性以及CP与LDL相互作用所需的CP的决定因素。目标2。研究CP在体内炎症过程中氧化中的作用。 CP-NULL小鼠将受到炎症的败血症和腹膜炎模型，并且将通过LC-MAS谱图测量腹膜灌洗和血浆中的特异性脂质和蛋白质氧化产物，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。