Mechanism of ABCA1-mediated CEC to lipidated HDL particles

摘要（项目2） 该项目资助的一个关键主题是ATP结合盒转运蛋白A1（ABCA 1）， 与无脂载脂蛋白A-I（APOA 1）和某些高密度脂蛋白（HDL）的特异性接触 亚种从细胞中输出脂质以产生成熟的HDL。从动脉壁清除胆固醇 预防心血管疾病。项目2接受了项目1的观察，即小HDL是一种 通过ABCA 1促进胆固醇流出的亚种，并且对于心脏保护特别重要， 糖尿病此外，我们发现HDL中含量第二丰富的蛋白质载脂蛋白A-II的存在， （APOA 2）也促进ABCA 1介导的胆固醇流出至完全脂化的HDL颗粒。这些 这些观察结果反驳了普遍接受的观点，即只有脂质贫乏的载脂蛋白才能与ABCA 1结合。 采取机械和功能的方法，项目2的主要目标是了解脂化如何 HDL的形式可以与ABCA 1相互作用以促进脂质流出。中心假设是，APOA 1在 小HDL，以及含有APOA 2的HDL，经历构象变化，可能在其C-末端， 这使得它能够与ABCA 1相互作用（称为“翻转末端假说”）。Aim 1将使用高分辨率的低温- EM和其他结构技术，以了解APOA 2如何影响APOA 1的结构和功能 在胆固醇流出试验中。我们将与项目3携手合作，不仅为APOA 1/A2相互作用建模， 还可以观察这些粒子如何与ABCA 1相互作用。所涉及的特定APOA 2序列将是 确定着眼于开发刺激胆固醇从ABCA 1转移到ABCA 1的治疗剂， 含脂质的HDL颗粒。目标2将使用APOA 1的新突变来改变HDL颗粒的大小。 沿着复杂的粒子重组和结构技术，如氢-氘 交换，以确定小HDL中允许其与ABCA 1接合的结构特征。我们还将确定 并在结构上表征人血浆中可与ABCA 1相互作用的HDL亚颗粒。Therapeutics 作为ABCA 1胆固醇流出底物的贫脂APOA 1的增强水平正在评估中， 临床试验我们的工作表明，设计出不仅能增加胆固醇， ABCA 1流出到脂质贫乏的受体，也流出到预先形成的HDL颗粒-一个容量高得多的池 因为它构成了循环APOA 1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.