Mechanism of ABCA1-mediated CEC to lipidated HDL particles

摘要（项目 2） 该计划项目拨款的一个关键主题是 ATP 结合盒转运蛋白 A1 (ABCA1) 与无脂载脂蛋白 A-I (APOA1) 和某些高密度脂蛋白 (HDL) 发生特异性接触 亚种从细胞输出脂质以产生成熟的高密度脂蛋白。从动脉壁去除胆固醇 预防心血管疾病。项目 2 继承了项目 1 的观察结果，即小型 HDL 是一种 通过 ABCA1 促进胆固醇流出的亚种，对于心脏保护尤其重要 糖尿病。此外，我们发现 HDL 的第二丰富蛋白质载脂蛋白 A-II 的存在 (APOA2)，还促进 ABCA1 介导的胆固醇流出至完全脂化的 HDL 颗粒。这些 观察结果反驳了普遍接受的观点，即只有低脂载脂蛋白才能与 ABCA1 结合。 采用机械和功能方法，项目 2 的主要目标是了解脂质如何 HDL 形式可以与 ABCA1 相互作用，促进脂质流出。中心假设是 APOA1 小 HDL 以及含有 APOA2 的 HDL 可能会在其 C 末端发生构象变化， 允许它与 ABCA1 相互作用（称为“翻转末端”假设）。目标 1 将使用高分辨率冷冻 使用 EM 和其他结构技术来了解 APOA2 如何影响 APOA1 的结构及其功能 在胆固醇流出测定中。我们将与项目 3 携手合作，不仅模拟 APOA1/A2 相互作用 还可以可视化这些粒子如何与 ABCA1 相互作用。涉及的具体 APOA2 序列将是 确定了开发刺激胆固醇从 ABCA1 转移到 含有脂质的HDL颗粒。目标 2 将使用 APOA1 的新突变来改变 HDL 颗粒的大小 以及复杂的粒子重组和结构技术，例如氢-氘 交换，以确定小 HDL 中允许其与 ABCA1 结合的结构特征。我们还将确定 并对人血浆中可与 ABCA1 相互作用的 HDL 亚颗粒进行结构表征。治疗学 正在评估提高贫脂 APOA1 水平作为 ABCA1 胆固醇流出底物的方法 临床试验。我们的工作表明，设计出不仅会增加胆固醇的疗法是可能的 ABCA1 流出至缺乏脂质的受体，但也流出至预先形成的 HDL 颗粒——一个具有更高容量的池 因为它占流通 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.