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

人类冠状动脉粥样硬化过早发生率 人口数量与高浓度浓度的降低高度相关 密度脂蛋白 (HDL) 及其主要脱辅基蛋白 Apo A-I 存在于 血。转基因和基因敲除动物研究最终表明 循环 HDL 的“保护作用”主要取决于其 独特的接受和组织胆固醇的能力。这也是一个函数 其激活卵磷脂酶的能力：胆固醇 胆固醇酰基转移酶 (LCAT) 胆固醇酰基转移酶 (LCAT) 胆固醇在血浆室中转化为胆固醇酯。的 胆固醇从动脉壁和外周的定向运动 组织朝向其唯一的分解代谢部位（活体）涉及许多 精心研究的步骤。 Apo-AI 似乎在以下方面发挥着关键作用： 每一步。 Apo A-I 是外排的主要受体 来自外周细胞的胆固醇。与磷脂、apo A-I 和 胆固醇形成新生盘状 HDL，这是首选底物 用于等离子体 LCAT。这种酶负责将新的 胆固醇外流为胆固醇酯。疏水性的积累 胆固醇酯作为球形 HDL 核心的脂滴及其 最终将胆固醇酯输送到活体中，完成了“逆转” 胆固醇转运”途径。在本研究提案中。我们会 研究“LCAT 酶激活的分子基础” 载脂蛋白A-I。已知这一重要的酶促途径存在缺陷 apo A-I 编码序列中携带某些突变的人类。 然而，目前尚不清楚apo A-I蛋白“如何”在apo表面 新生盘状 HDL 颗粒共同激活这一催化过程。 因此，为了阐明这一过程的分子机制，我们将 使用PCR诱变构建一系列特定的氨基酸突变体， 然后使用我们的杆状病毒生产毫克量的这些蛋白质 Sf-9细胞系统。突变的apo A-I蛋白将被广泛研究 使用生物化学和生物物理技术来确定哪个关键 结构特征负责新生 HDL 的正确定向 用于 LCAT 催化的磷脂酰基链。

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.