ABCA1-mediated biogenesis of nascent HDL particles

描述(申请人提供)：高密度脂蛋白胆固醇(高密度脂蛋白胆固醇)是一种特别有希望的冠心病(CHD)治疗干预候选药物。尽管高密度脂蛋白-c的绝对水平与冠心病风险呈负相关，但越来越多的证据表明，高密度脂蛋白颗粒中的胆固醇从周围组织流向肝脏--即反向胆固醇运输(RCT)--是一个更重要的动脉粥样硬化保护因素。最近的研究表明，不同的高密度脂蛋白颗粒种类对RCT的刺激程度不同。颗粒不均匀的起源尚不清楚。在新生的高密度脂蛋白中，一些粒子的形态已经很明显。新生高密度脂蛋白是由细胞脂质和细胞外载脂蛋白AI(ApoAI)通过三磷酸腺苷结合盒转运体A1(ABCA1)介导的过程组装而成。在具体目标1中，我们提出了一种假设，即ABCA1在质膜不同微环境中的定位是导致新生的高密度脂蛋白颗粒异质性的原因。质膜的脂质组成和ABCA1可能定位于不同的质膜结构域将被操纵，以确定这些操纵对新生的高密度脂蛋白群体产生什么影响。ApoAI结合保护ABCA1免受降解，并在前馈调节环中促进新生高密度脂蛋白颗粒的形成。在特定的目标2中，我们提出了两种主要的方法来确定ABCA1上可能的载脂蛋白AI结合位点。在一种方法中，ABCA1和ApoAI将被交联，然后ABCA1-ApoAI复合体将被用质谱学分析。在第二种方法中，通过计算选择的候选ABCA1区域将被化学合成，并使用表面等离子体共振测试与ApoAI的结合。用这两种方法确定的ABCA1区域将使用突变分析和结合完成分析进行验证。从这个项目中获得的知识将有助于设计新的疗法来刺激RCT，并最大限度地产生最具动脉粥样硬化保护作用的高密度脂蛋白。 公共卫生相关性：冠心病是美国和世界主要的死亡原因。目前专注于降低“坏”(低密度脂蛋白)胆固醇水平的治疗方法可以减少但不能完全消除冠心病的发生。这项拟议的研究应该有助于开发补充疗法，利用心脏病降低“好”(高密度脂蛋白)胆固醇的特性。

DESCRIPTION (provided by applicant): High-density lipoprotein cholesterol (HDL-c) is an especially promising candidate for therapeutic intervention against coronary heart disease (CHD). Although, absolute levels of HDL-c correlate inversely with the CHD risk, there is growing evidence that cholesterol flux in HDL particles from peripheral tissues to the liver - i.e., reverse cholesterol transport (RCT) - is a more important atheroprotective factor. Recent research shows that different HDL particle species stimulate RCT to varying extents. The origins of particle heterogeneity are unclear. Some particle speciation is already evident in nascent HDL. Nascent HDL is assembled from cellular lipids and extracellular apolipoprotein AI (apoAI) through a process mediated by ATP-binding cassette transporter A1 (ABCA1). In Specific Aim 1, we propose to test the hypothesis that localization of ABCA1 in different microenvironments of the plasma membrane is responsible for nascent HDL particle heterogeneity. Lipid composition of the plasma membrane and putative localization of ABCA1 to different plasma membrane domains will be manipulated to determine what effects these manipulations exert on the nascent HDL population. ApoAI binding protects ABCA1 from degradation and promotes nascent HDL particle formation in a feed-forward regulatory loop. In Specific Aim 2, we propose two primary approaches to identify putative apoAI binding sites on ABCA1. In one approach, ABCA1 and apoAI will be cross-linked and then ABCA1-apoAI complexes will be analyzed using mass spectrometry. In the second approach, computationally selected candidate ABCA1 regions will be chemically synthesized and tested for binding to apoAI using surface plasmon resonance. The regions of ABCA1 identified with the two approaches will be validated using mutagenic analyses and binding completion assays. The knowledge gained from this project will aid in design of novel therapies to stimulate RCT and maximize production of the most atheroprotective HDL species. PUBLIC HEALTH RELEVANCE: Coronary heart disease is a leading cause of death in the US and the world. Present therapies that focus on lowering levels of "bad" (LDL) cholesterol reduce but do not completely eliminate coronary heart disease occurrences. The proposed research should facilitate development of supplemental therapies that exploit heart disease reducing properties of "good" (HDL) cholesterol.