Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

项目摘要 清道夫受体B I型（SR-BI）和HDL的受体-配体复合物负责胆固醇 胆固醇通过胆固醇逆向转运（RCT）从体内排出，在预防动脉粥样硬化中至关重要。 我们研究的长期目标是了解调节SR-BI介导的 胆固醇酯（CE）从HDL递送到肝脏用于排泄。这项申请的科学前提是 是基于越来越多的文献，表明心血管疾病的风险可以降低的战略，促进 通过增强胆固醇流出和RCT清除胆固醇。前提也得到突变的支持 在高HDL-C患者中发现的SCARB 1（人类SR-BI基因）中， HDL-CE和增加CVD的风险。虽然HDL-C的有效清除取决于RCT的最后步骤， 关于触发HDL-CE选择性摄取的SR-BI/HDL相互作用的重要性的研究仍然有限。 在上一个资助周期中，我们最大的发现是第一个高分辨率的核磁共振结构， SR-BI包含C-末端跨膜（TM）结构域并有助于SR-BI寡聚化， 除了含有具有独特疏水性质的短α螺旋的相邻细胞外区域之外。 在这个应用中，我们建立在这些令人兴奋的新发现和额外的有希望的初步数据，以测试 总体假设，通过RCT的胆固醇通量是由SR-BI的结构特征驱动的，这些结构特征对于 膜结合和受体寡聚化。在目标1中，我们将使用结构指导的诱变和 一系列创新的生物物理技术，以确定是否受体/膜相互作用涉及 推测的细胞外质膜螺旋是介导SR-BI的胆固醇转运功能所必需的。 接下来，我们将通过评估小鼠巨噬细胞-粪便随机对照试验来测试巨噬细胞膜螺旋在体内的重要性 表达突变SR-BI受体，其中螺旋疏水性已经改变。在目标2中，我们将使用尖端的 核磁共振策略和顺磁弛豫实验，以及结构导向诱变，以确定 SR-BI低聚物的组织，一种被认为是胆固醇从HDL中转移所必需的复合物 到细胞。此外，我们将映射SR-BI的TM结构域之间的精确结合界面， 介导HDL-CE选择性摄取进入质膜。最后，在目标3中，使用非 SR-BI寡聚突变体和相关对照，我们还将进行巨噬细胞-粪便RCT研究， 作为动脉粥样硬化研究，以确定SR-BI寡聚化在鼠模型中的体内功能相关性。 体外和体内研究的结合使用，以及创新和最先进的技术，将 推进我们对SR-BI分子结构的了解。重要的是，我们的研究结果将 确定旨在预防高胆固醇血症及其相关病理的未来疗法， 动脉粥样硬化，通过精确定位驱动SR-BI介导的选择性摄取的潜在结构机制， HDL-CE和体内净胆固醇排泄。

PROJECT SUMMARY The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and HDL is responsible for cholesterol disposal from the body via reverse cholesterol transport (RCT) and is critical in the prevention of atherosclerosis. The long-term objective of our research is to understand the mechanisms that regulate SR-BI-mediated delivery of cholesteryl ester (CE) from HDL to the liver for excretion. The scientific premise of this application is based on a growing body of literature that suggests CVD risk can be reduced by strategies that promote cholesterol clearance through enhanced cholesterol efflux and RCT. The premise is also supported by mutations in SCARB1 (the human SR-BI gene), identified in patients with high HDL-C, that prevent the selective uptake of HDL-CE and increase the risk of CVD. While efficient clearance of HDL-C hinges on the last steps of RCT, studies on the importance of the SR-BI/HDL interaction that triggers selective uptake of HDL-CE remain limited. In the previous funding cycle, our biggest discovery was the first high-resolution NMR structure of a region of SR-BI that encompasses the C-terminal transmembrane (TM) domain and contributes to SR-BI oligomerization, in addition to an adjacent extracellular region that harbors a short alpha helix with unique hydrophobic properties. In this application, we build on these exciting novel findings and additional promising preliminary data to test the overall hypothesis that cholesterol flux via RCT is driven by structural features of SR-BI that are important for membrane association and receptor oligomerization. In Aim 1, we will use structure-guided mutagenesis and a series of innovative biophysical techniques to determine whether receptor/membrane interactions involving a putative extracellular juxtamembrane helix are required to mediate the cholesterol transport functions of SR-BI. Next, we will test the in vivo importance of the juxtamembrane helix by assessing macrophage-to-feces RCT in mice expressing mutant SR-BI receptors where helix hydrophobicity has been altered. In Aim 2, we will use cutting-edge NMR strategies and paramagnetic relaxation experiments, as well as structure-guided mutagenesis, to identify the organization of the SR-BI oligomer, a complex deemed essential for the movement of cholesterol from HDL to cells. Further, we will map the precise binding interfaces between TM domains of SR-BI that are likely critical in mediating the selective uptake of HDL-CE into the plasma membrane. Finally, in Aim 3, using a non- oligomerizing mutant of SR-BI and relevant controls, we will perform macrophage-to-feces RCT studies, as well as atherosclerosis studies, to define the in vivo functional relevance of SR-BI oligomerization in murine models. The combined use in vitro and in vivo studies, together with innovative and state-of-the-art technologies, will advance our knowledge of the molecular architecture of SR-BI. Importantly, the outcomes of our studies will identify future therapies aimed at preventing hypercholesterolemia and its associated pathologies such as atherosclerosis, by pinpointing the underlying structural mechanisms that drive SR-BI-mediated selective uptake of HDL-CE and net cholesterol excretion from the body.