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

早期冠状动脉粥样硬化在人类中的发生率 人口与高污染浓度的降低高度相关 密度脂蛋白(HDL)及其主要载脂蛋白apo A-I 血。转基因和基因敲除动物研究已经确凿地表明 循环中高密度脂蛋白的“保护作用”主要是由其功能决定的。 独特的接受和组织胆固醇的能力。它也是一个函数 其激活卵磷脂酶的能力：胆固醇 胆固醇酰基转移酶(LCAT) 血浆中胆固醇到胆固醇酯的转化。这个 胆固醇从动脉壁和外周的定向移动 组织朝向其唯一分解代谢的部位，即肝脏，涉及许多 经过深思熟虑的步骤。载脂蛋白-AI似乎在 这些步骤中的每一个。载脂蛋白A-I是外排的主要受体 外周细胞中的胆固醇。与磷脂、载脂蛋白A-I和 胆固醇形成新生的盘状高密度脂蛋白，这是首选的底物 用于血浆LCAT。这种酶负责将新的 将胆固醇排出为胆固醇酯。疏水性物质的积累 胆固醇酯作为球形高密度脂蛋白核心的脂滴及其应用 最终将胆固醇酯输送到活着的人身上完成了这一反转 胆固醇转运途径。在这项研究中，我们将 探讨LCAT酶激活的分子基础 载脂蛋白A-I。这一重要的酶途径是已知存在缺陷的 在载脂蛋白A-I编码序列中携带某些突变的人类。 然而，目前尚不清楚载脂蛋白A-I蛋白是如何在 新生的盘状高密度脂蛋白颗粒共同激活了这一催化过程。 因此，为了阐明这一过程的分子机制，我们将 利用聚合酶链式反应诱变技术构建了一系列特异的氨基酸突变体， 然后用我们的杆状病毒生产毫克量的这些蛋白质 SF-9细胞系。突变的载脂蛋白A-I将被广泛研究 使用生化和生物物理技术来确定 结构特征负责正确定位新生的高密度脂蛋白 用于LCAT催化的磷脂酰链。

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.