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

APO A-1 构象与颗粒 L 大小之间的结构关系

• 批准号: 7537462
• 负责人: Mary G Sorci-Thomas
• 金额: $ 25.74万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7537462
• 关键词: 3-Dimensional; ATP-Binding Cassette Transporters; Address; Affinity; Amino Acid Substitution; Antiatherogenic; Apolipoproteins; Apolipoproteins A; Atherosclerosis; Binding; Biological; Blood Circulation; C-terminal; Catabolism; Cell Culture System; Cell membrane; Cells; Cessation of life; Cholesterol; Cholesterol Homeostasis; Chronic; Complex; Condition; Consumption; Coronary Occlusions; Cysteine; Data; Development; Dietary Fats; Disease; Dissociation; Disulfides; Dominant-Negative Mutation; Enzymes; Equilibrium; Excretory function; Helix (Snails); High Density Lipoproteins; Human; Incubated; Individual; Inflammatory; Investigation; Length; Link; Lipid Binding; Lipids; Lipoproteins; Liver; Maintenance; Measurement; Measures; Mediating; Modeling; Molecular Conformation; Mus; Mutation; Numbers; Particle Size; Phenotype; Phospholipids; Plasma; Principal Investigator; Production; Property; Proteins; Rate; Recombinants; Repression; Role; Series; Sodium Cholate; Structure; Techniques; Testing; Time; Transgenic Mice; Transgenic Organisms; X-Ray Crystallography; base; cysteinylcysteine; diadenosine pyrophosphate; ear helix; in vivo; metabolic abnormality assessment; mouse model; mutant; particle; prevent; programs; response; size

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.

动脉粥样硬化是一种慢性炎症性疾病，其由食用脂肪促进， 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 通过血浆apoA-1（高密度脂蛋白（HDL）的主要蛋白质成分）的存在介导。 HDL apoA-I在全身胆固醇稳态中的作用已被广泛研究，其作用是调节胆固醇水平。 是将胆固醇从外周引导到肝脏排泄。尽管进行了深入的调查， 关于apoA-l调节细胞胆固醇水平的机制以及如何调节细胞胆固醇水平的问题仍然存在。 apoprotien的独特结构特征促进胆固醇转运。为了更全面地了解 为了阐明apoA-I在胆固醇转运中作用的结构基础，我们将进行三个具体的目标， 通过ABCA 1形成新生HDL所必需的结构重组。在目标1中，我们建议 通过确定apoA-I在四个表面上的构象来解决apoA-I在胆固醇稳态中的作用， 不同大小的ABCA 1亚类产生新生HDL。同样作为目标1的一部分，我们将研究 无脂质的apoA-1通过其从ABCA 1获得脂质的“解折叠”步骤。为此，我们将构建一个 一系列“栓系的”二硫化物apoA-I突变体，其将阻止关键的中间解折叠步骤， 4-螺旋束在组织和结合磷脂和胆固醇时必须转变。在Aim 2中，我们 将研究野生型apoA-1 HDL显性负抑制的机制基础， 通过研究螺旋6突变体L159 R的脂化作用， apoA-1，ABCA 1。我们的初步研究表明，这个单氨基酸取代突变体，L159 R， apoA-I与野生型apoA-I竞争磷脂和胆固醇，导致apoA-I的表达降低。 在模型细胞培养系统中ABCA 1对野生型apoA-1的总体脂化。在目标3中，我们计划确定 与L159 R apoA-1突变体相关的显性阴性表型是促动脉粥样硬化还是抗动脉粥样硬化 在高脂血症小鼠中。使用表达野生型或L159 R apoA-1的转基因小鼠，我们将 确定突变体apoA-1对动脉粥样硬化发展的保护程度，以及 作为研究血浆中低浓度L159 R apoA-1的体内基础，因为它与 显性阴性表型