Structural Relationship Between APO A-1 Comformation and the Extent of Particle L

动脉粥样硬化是一种慢性炎症性疾病，由饮食脂肪和 胆固醇。动脉粥样硬化进展到导致冠状动脉闭塞和/或死亡的程度是 高密度脂蛋白的主要蛋白质成分--载脂蛋白A-L的存在。 高密度脂蛋白载脂蛋白A-L在全身胆固醇稳态中的作用已被广泛研究及其作用 就是将胆固醇从外周引导到肝脏排泄。尽管进行了密集的调查，但主要的 关于载脂蛋白A-L调节细胞胆固醇水平的机制以及 载脂蛋白的独特结构特征促进了胆固醇的运输。为了更全面地了解 载脂蛋白A-L在胆固醇转运中作用的结构基础，我们将执行三个具体目标，以澄清 通过ABCA1形成新生高密度脂蛋白所必需的结构重组。在目标1中，我们建议 通过测定载脂蛋白A-L在胆固醇动态平衡中的构象研究载脂蛋白A-L 不同大小的ABCA1亚类产生了新生的高密度脂蛋白。同样作为目标1的一部分，我们将研究 无脂载脂蛋白A-L从ABCA1获得脂类的步骤展开。为此，我们将构建一个 一系列‘拴系’二硫化物载脂蛋白A-L突变体，将阻止关键的中间步骤展开，通过 4-螺旋束在组织和结合磷脂和胆固醇时必须过渡。在目标2中，我们 将探讨野生型载脂蛋白A-L高密度脂蛋白显性负抑制的机制基础 通过研究螺旋6突变体L159R的脂化，在携带这种突变的人类中观察到 载脂蛋白A-L由ABCA1。我们的初步研究表明，该单一氨基酸替代突变体L159R 载脂蛋白A-L与野生型载脂蛋白A-L竞争磷脂和胆固醇，导致 模型细胞培养系统中ABCA1对野生型载脂蛋白A-L的全面脂化作用。在目标3中，我们计划确定 L159R载脂蛋白A-L突变体相关的显性负性表型是促动脉粥样硬化还是抗动脉粥样硬化 在高脂血症小鼠身上。使用表达野生型或L159R载脂蛋白A-L的转基因小鼠，我们将 确定突变的载脂蛋白A-L对动脉粥样硬化发展的保护程度 L159R载脂蛋白A-L血浆低浓度的体内基础研究 显性负性表型。

Atherosclerosis is a chronic inflammatory disease that is promoted by the consumption of dietary fat and cholesterol. The extent to which atherosclerosis progresses to cause coronary occlusion and/or death is mediated by the presence of plasma apoA-l, the main protein constituent of high density lipoproteins (HDL). The role of HDL apoA-l in whole body cholesterol homeostasis has been extensively investigated and its role is to direct cholesterol from the periphery to the liver for excretion. Despite intensive investigations, major questions remain regarding the mechanism by which apoA-l regulates cellular cholesterol levels and how the apoprotien's unique structural features facilitate cholesterol transport. To more completely understand the structural basis for apoA-l's role in cholesterol transport, we will carry out three specific aims that will clarify the structural reorganization necessary for the formation of nascent HDL via ABCA1. In Aim 1 we propose to address the role of apoA-l in cholesterol homeostasis by determining the conformation of apoA-l on four different sized subclasses of ABCA1 generated nascent HDL. Also as part of Aim 1, we will examine the 'unfolding' steps' through which lipid-free apoA-l acquires lipid from ABCA1. To do this we will construct a series of 'tethered' disulfide apoA-l mutants that will prevent key intermediate unfolding steps through which the 4-helix bundle must transition as it organizes and binds phospholipid and cholesterol. In the Aim 2, we will investigate the mechanistic basis for the dominant negative repression of wild-type apoA-l HDL, as observed in humans who carry this mutation, by investigating the lipidation of the helix 6 mutant, L159R apoA-l by ABCA1. Our preliminary studies suggest that this single amino acid substitution mutant, L159R apoA-l, competes with wild-type apoA-l for phospholipid and cholesterol, resulting in a reduction in the overall lipidation of wild-type apoA-l by ABCA1 in a model cell culture system. In Aim 3, we plan to determine whether the dominant negative phenotype associated with the L159R apoA-l mutant is pro- or antiatherogenic in hyperlipidemic mice. Using transgenic mice that express wild-type or L159R apoA-l, we will determine the extent to which the mutant apoA-l protects against the development of atherosclerosis, as well as investigating the in vivo basis for the low concentration of L159R apoA-l in plasma as it relates to the dominant negative phenotype.