IMMUNOCHEMICAL STRUCTURE/FUNCTION OF APOLIPOPROTEIN AI

该提案概述了经典的蛋白质结构/功能研究。它 测试蛋白质的特定结构域可以是 标识了授予特定函数，并且该函数可以在 特定的情况下，被该蛋白质的较小片段所模仿。 这些研究将在重要的血浆蛋白上进行， 载脂蛋白(Apo)A-I。载脂蛋白A-I是血浆高密度脂蛋白的主要载脂蛋白 密度脂蛋白(HDL)。血液循环水平高的个体 高密度脂蛋白患动脉粥样硬化并发症的风险较低。多重 高密度脂蛋白的功能有助于其有益的特性。阿波A-I 通过促进胆固醇外流促进胆固醇的反向转运 从动脉粥样硬化病变的泡沫细胞和激活血浆 酯化酶，卵磷脂：胆固醇酰基转移酶(LCAT)。 这个重要的辅基蛋白的结构/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 这将利用一组28个独特的人类载脂蛋白A-I特异性单抗 抗体和85个纯化的合成肽，以及一个结构突变体 将研究超过15种基因工程和表达的方法 人类载脂蛋白A-I的突变形式。第一个目标是检验这样一个假设 载脂蛋白A-I发生构象变化，无脂载脂蛋白 组装到含有脂质的圆盘或球体上。因为X射线 结晶学不能用来解释载脂蛋白A-I的结构 它与脂质有关，免疫化学方法可以提供这一点 否则就无法获得信息。第二个目标将测试 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胆固醇外流和 激活LCAT。同样，将确定载脂蛋白A-I的突变形式 它们已经改变了调节细胞胆固醇外流或行为的能力 作为LCAT的辅助因子。在第三和第四个目标中，关键多肽将 在体外测试它们模拟载脂蛋白A-I功能的能力 分别在活体内。这些研究的成功完成将 提供：可代谢鉴定和分离的抗体试剂 不同的高密度脂蛋白亚群，关于结构的分子信息 载脂蛋白A-I的防护功能特性要求，以及 体内外模拟载脂蛋白A-I特异性功能的多肽 载脂蛋白A-I的有益作用可用于治疗 动脉硬化。

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.