Mechanism of ABCA1-mediated CEC to lipidated HDL particles

摘要(项目2) 这项计划项目拨款的一个关键主题是ATP结合盒式磁带传送器A1(ABCA1)使 与无脂载脂蛋白A-I(APOA1)和某些高密度脂蛋白(HDL)的特异性接触 从细胞中输出脂质以产生成熟的高密度脂蛋白的亚种。动脉壁中胆固醇的去除 预防心血管疾病。项目2从项目1开始，S观察到小型HDL是一种 通过ABCA1促进胆固醇外流的亚种，它对心脏保护尤其重要 糖尿病。此外，我们发现，高密度脂蛋白第二丰富的蛋白质，载脂蛋白A-II的存在 (APOA2)，也促进ABCA1介导的胆固醇外流到完全脂化的高密度脂蛋白颗粒。这些 观察结果与普遍接受的观点相反，即只有缺乏脂质的载脂蛋白才能与ABCA1结合。 采用机械和功能的方法，项目2的主要目标将是了解如何脂肪 不同形式的高密度脂蛋白可以与ABCA1相互作用，促进脂质外流。中心假设是APOA1中的 小的HDL和那些含有APOA2的HDL经历了构象变化，可能在其C-末端， 这使得它能够与ABCA1相互作用(称为“翻转末端”假说)。目标1号将使用高分辨率低温- EM等结构技术研究载脂蛋白A_2对载脂蛋白A_2 S结构及其功能的影响 在胆固醇外流分析中。我们将与项目3携手合作，不仅为APOA1/A2交互建模 也是为了可视化这些粒子如何与ABCA1相互作用。具体涉及的APOA2序列如下 被认为着眼于开发刺激胆固醇从ABCA1转移到 含脂高密度脂蛋白颗粒。目标2将使用APOA1的新突变来改变高密度脂蛋白颗粒的大小 以及先进的粒子重组和结构技术，如氢-重氢 Exchange，以确定小型高密度脂蛋白中允许其与ABCA1接触的结构特征。我们还将确定 并从结构上表征了人类血浆中能与ABCA1相互作用的HDL亚粒子。治疗学 作为ABCA1胆固醇外流底物的低脂载脂蛋白A1水平的提高正在进行评估 临床试验。我们的工作表明，设计不仅可以增加胆固醇的疗法是可能的。 ABCA1外流到缺乏脂质的受体，但也外流到预先形成的高密度脂蛋白颗粒-一个容量大得多的池 因为它构成了流通中APOA1的最大份额。

SUMMARY (Project 2) A key theme of this Program Project Grant is that ATP-binding cassette transporter A1 (ABCA1) makes specific contacts with both lipid-free apolipoprotein A-I (APOA1) and certain high-density lipoprotein (HDL) subspecies to export lipids from cells to generate mature HDL. This removal of cholesterol from the artery wall protects against cardiovascular disease. Project 2 picks up on Project 1’s observation that small HDLs are a subspecies that promotes cholesterol efflux via ABCA1 and that it is especially important for cardioprotection in diabetes. In addition, we found that the presence of HDL’s second-most abundant protein, apolipoprotein A-II (APOA2), also promotes ABCA1-mediated cholesterol efflux to fully lipidated HDL particles. These observations counter the commonly accepted idea that only lipid-poor apolipoproteins can engage ABCA1. Taking a mechanistic and functional approach, the main goal of Project 2 will be to understand how lipidated forms of HDL can interact with ABCA1 to promote lipid efflux. The central hypothesis is that the APOA1 in small HDLs, and in those containing APOA2, undergoes a conformational change, possibly in its C-terminus, that allows it to interact with ABCA1 (called the “Flipped Ends’ hypothesis). Aim 1 will use high-resolution cryo- EM and other structural techniques to understand how APOA2 affects APOA1’s structure and its functionality in cholesterol efflux assays. We will work hand-in-hand with Project 3 to not only model APOA1/A2 interactions but also to visualize how those particles interact with ABCA1. The specific APOA2 sequences involved will be identified with an eye toward developing therapeutics that stimulate the transfer of cholesterol from ABCA1 to lipid-containing HDL particles. Aim 2 will use novel mutations of APOA1 that alter the sizes of HDL particles along with sophisticated particle reconstitution and structural techniques, such as hydrogen-deuterium exchange, to identify structural features in small HDL that allow it to engage with ABCA1. We will also identify and structurally characterize HDL subparticles in human plasma that can interact with ABCA1. Therapeutics that boost levels of lipid-poor APOA1 as substrates for cholesterol efflux by ABCA1 are being evaluated in clinical trials. Our work suggests that it may be possible to design therapies that not only increase cholesterol efflux by ABCA1 to lipid-poor acceptors but also to preformed HDL particles—a pool with much higher capacity as it comprises the lion’s share of circulating APOA1.