STRUCTURE/FUNCTION RELATIONSHIPS OF APOLIPOPROTEIN A (APOA-1)

载脂蛋白 A (APOA-1) 的结构/功能关系

• 批准号: 6338878
• 负责人: Mary G Sorci-Thomas
• 金额: $ 19.92万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6338878
• 关键词: CHO cells; apolipoproteins; atherosclerosis; blood lipoprotein metabolism; chemical binding; cholesterol esters; circular dichroism; enzyme activity; enzyme induction /repression; enzyme mechanism; esterification; fluorescence spectrometry; gene mutation; lipid structure; phosphatidylcholine sterol acyltransferase; protein structure function; site directed mutagenesis; structural biology

The incidence of premature coronary atherosclerosis in the human population is highly correlated to decreased concentrations of high density lipoprotein (HDL) and its major apoprotein, apo A-I found in the blood. Transgenic and knockout animal studies have shown conclusively that the "protective effect" of circulating HDL is primarily a function of its unique ability to accept and organize cholesterol. It is also a function of its ability to activate the enzyme lecithin: cholesterol acyltransferase (LCAT) for cholesterol acyltransferase (LCAT) for cholesterol to cholesterol ester conversion in the plasma compartment. The directional movement of cholesterol from the artery wall and peripheral tissues towards its only site of catabolism, the live, involves a number of well studied steps. Apo-AI appears to be to be plays a key role in each of these steps. Apo A-I is the primary acceptor for effluxed cholesterol from peripheral cells. Together with phospholipid, apo A-I and cholesterol form nascent discoidal HDL which is the preferred substrate for the plasma LCAT. This enzyme is responsible for converting newly effluxed cholesterol to cholesterol ester. Accumulation of the hydrophobic cholesterol ester as a lipid droplet in the core of spherical HDL and its ultimate delivery of cholesterol ester to the live completes the "reverse cholesterol transport" pathway. In this research proposal. we will investigate the molecular basis for the "activation of the enzyme LCAT by apo A-I. This important enzymatic pathway is known to be defective in humans who carry certain mutations within the apo A-I coding sequence. However, it is not known "how" the apo A-I protein on the surface of a nascent discoidal HDL particle co-activate this catalytic process. Therefore, to elucidate the molecular mechanism of this process we will construct a series of specific amino acid mutants using PCR mutagenesis, then produce these proteins in milligram quantities using our baculoviral Sf-9 cell system. The mutant apo A-I proteins will be extensively studied using both biochemical and biophysical techniques to determine which key structural features are responsible for properly orienting the nascent HDL phospholipid acyl chain for LCAT catalysis.

人早发冠状动脉粥样硬化的发病率 人口数量与高浓度 密度脂蛋白（HDL）及其主要载脂蛋白，载脂蛋白A-I，发现在 血转基因和基因敲除动物研究已经明确表明， 循环HDL的“保护作用”主要是其 接受和组织胆固醇的独特能力。也是一种功能 它能够激活酶卵磷脂：胆固醇 胆固醇酰基转移酶（LCAT） 血浆室中胆固醇向胆固醇酯的转化。的 胆固醇从动脉壁和外周的定向运动 组织朝向其唯一的catenorism部位，即肝脏，涉及许多 研究的步骤。Apo-AI似乎在以下方面起着关键作用： 每一个步骤。载脂蛋白A-I是外排的主要受体， 胆固醇从外周细胞。与磷脂、载脂蛋白A-I和 胆固醇形成新生盘状HDL，其是优选底物 等离子LCAT这种酶负责将新的 将胆固醇流出为胆固醇酯。疏水物的积累 胆固醇酯作为球状HDL核心的脂滴， 胆固醇酯最终输送到肝脏完成了“逆转 胆固醇转运”途径。在这项研究计划中。我们将 研究“LCAT酶的激活”的分子基础， 载脂蛋白A-I已知这种重要的酶途径在以下方面存在缺陷： 在apo A-I编码序列内携带某些突变的人。 然而，目前尚不清楚载脂蛋白A-I蛋白是“如何”在细胞表面表达的。 新生盘状HDL颗粒共同激活该催化过程。 因此，为了阐明这一过程的分子机制，我们将 利用PCR诱变构建一系列特异性氨基酸突变体， 然后用我们的杆状病毒 Sf-9细胞系统。突变型载脂蛋白A-I蛋白将被广泛研究 利用生物化学和生物物理技术来确定 结构特征负责正确定向新生HDL 用于LCAT催化的磷脂酰基链。