IMMUNOCHEMICAL STRUCTURE/FUNCTION OF APOLIPOPROTEIN AI

载脂蛋白 AI 的免疫化学结构/功能

• 批准号: 2702190
• 负责人: Linda K Curtiss
• 金额: $ 33.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702190
• 关键词: acyltransferase; apolipoproteins; chimeric proteins; cholesterol; clinical research; gene deletion mutation; human subject; laboratory mouse; laboratory rabbit; laboratory rat; macrophage; monoclonal antibody; phospholipids; protein structure function; synthetic peptide

This proposal outlines classical protein structure/function studies. It tests the hypotheses that specific structural domains of a protein can be identified that confer a specific function, and that this function can, in specific instances, be mimicked by a smaller fragment of that protein. These studies will be performed on the important plasma protein, apolipoprotein (apo) A-I. Apo A-I is the major apoprotein of plasma high density lipoproteins (HDL). Individuals with high levels of circulating HDL are at lower risk for the complications of atherosclerosis. Multiple functions of HDL contribute to its beneficial properties. Apo A-I promotes reverse cholesterol transport by facilitating cholesterol efflux from foam cells of the atherosclerotic lesion and activating the plasma esterifying enzyme, lecithin:cholesterol acyltransferase (LCAT). Structure/function relationships of this important apoprotein will be examined using two complementary approaches: an immunochemical approach that will utilize a panel of 28 unique human apo A-I-specific monoclonal antibodies and 85 purified synthetic peptides, and a structural mutant approach that will study over 15 genetically engineered and expressed mutant forms of human apo A-I. The first aim tests the hypothesis that conformational changes occur in apo A-I as the lipid-free apoprotein is assembled onto lipid-containing discs or spheres. Because X-ray crystallography cannot be used to elucidate the structure of apo A-I when it is associated with lipid, an immunochemical approach can provide this otherwise unobtainable information. The second aim will test the hypothesis that apo A-I contains specific functional domains. Antibodies will be identified that modulate cholesterol efflux from macrophages and activation of LCAT. Likewise, mutant forms of apo A-I will be identified that have altered ability to modulate cellular cholesterol efflux or act as a cofactor for LCAT. In the third and fourth aims, key peptides will be tested for their ability to mimic these apo A-I functions in vitro and in vivo, respectively. The successful completion of these studies will provide: antibody reagents that can identify and isolate metabolically distinct HDL subpopulations, molecular information about the structural requirements for the protective functional properties of apo A-I, and peptides that mimic specific functions of apo A-I in vitro and in vivo so that the beneficial role of apo A-I can be exploited for the treatment of atherosclerosis.

该建议概述了经典蛋白质结构/功能研究。 它 测试假设蛋白质的特定结构结构域可以是 确定赋予特定函数，并且此功能可以在 特定的实例，被该蛋白质的较小片段模仿。 这些研究将在重要的血浆蛋白上进行 载脂蛋白（APO）A-I。 apo a-i是血浆高的主要顶蛋白 密度脂蛋白（HDL）。 循环高水平的人 HDL的动脉粥样硬化并发症的风险较低。 多种的 HDL的功能有助于其有益特性。 apo a-i 通过促进胆固醇外排来促进反向胆固醇的转运 来自动脉粥样硬化病变的泡沫细胞并激活血浆 酯化酶，卵磷脂：胆固醇酰基转移酶（LCAT）。 这种重要的载蛋白的结构/功能关系将是 使用两种互补方法检查：一种免疫化学方法 这将利用一个由28个独特的人a-a-i特异性单克隆的面板 抗体和85种纯化的合成肽和结构突变体 将研究超过15种基因工程和表达的方法 人apo a-i的突变形式。 第一个目的检验了以下假设 apo A-1中发生构象变化，因为无脂肪蛋白是 组装到含脂质的圆盘或球体上。 因为X射线 晶体学不能用于阐明Apo A-I的结构 它与脂质有关，一种免疫化学方法可以提供 否则无法获得的信息。 第二个目标将测试 Apo A-I包含特定功能域的假设。 抗体 将确定从巨噬细胞调节胆固醇外排和 LCAT的激活。 同样，将确定apo a-i的突变形式 调节细胞胆固醇外排或作用的能力改变了 作为LCAT的辅助因子。 在第三和第四目标中，关键肽将 测试它们在体外模仿这些apo a-i功能的能力和 分别在体内。 这些研究的成功完成将 提供：可以代谢识别和分离的抗体试剂 独特的HDL亚群，有关结构的分子信息 APO A-I的保护功能特性的要求，以及 模仿Apo a-i在体外和体内的肽 可以利用Apo A-I的有益作用来处理 动脉粥样硬化。