IMMUNOCHEMICAL STRUCTURE/FUNCTION OF APOLIPOPROTEIN AI

该建议概述了经典的蛋白质结构/功能研究。 它 测试了蛋白质的特定结构域可以被 确定赋予特定功能，并且该功能可以， 在特定的情况下，可以被该蛋白质的较小片段模仿。 这些研究将对重要的血浆蛋白进行， 载脂蛋白A-I。 载脂蛋白A-I是高脂血症的主要载脂蛋白， 密度脂蛋白（HDL）。 高水平循环的个体 HDL是动脉粥样硬化并发症的低风险。 多 HDL的功能有助于其有益的特性。 APO a-I 通过促进胆固醇流出促进胆固醇逆向转运 从动脉粥样硬化病变的泡沫细胞中提取并激活血浆 卵磷脂：胆固醇酰基转移酶（LCAT）。 这一重要的载脂蛋白的结构/功能关系将是 使用两种互补的方法进行检查：免疫化学方法 将利用一组28种独特人apoA-I特异性单克隆抗体 抗体和85个纯化的合成肽，以及结构突变体 该方法将研究超过15种基因工程和表达 人载脂蛋白A-I的突变形式。 第一个目标是检验假设， apoA-I的构象发生变化， 组装到含脂质的盘或球体上。 因为X光 晶体学不能用来阐明apoA-I的结构， 它与脂质有关，免疫化学方法可以提供这一点， 否则无法获得的信息。 第二个目标将考验 假设载脂蛋白A-I含有特定的功能结构域。 抗体 将被鉴定为调节巨噬细胞的胆固醇流出， 激活LCAT。 同样，将鉴定载脂蛋白A-I的突变形式， 改变了调节细胞胆固醇流出的能力， 作为LCAT的辅助因子 在第三和第四个目标中，关键肽将 测试它们在体外模拟这些apo A-I功能的能力， 在体内，分别。 这些研究的成功完成将 提供：可代谢识别和分离的抗体试剂 不同的HDL亚群，关于结构的分子信息， 载脂蛋白A-I的保护功能特性要求，以及 在体外和体内模拟apo A-I的特异性功能的肽， apo A-I的有益作用可用于治疗 动脉粥样硬化

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.