Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

项目总结 清道夫受体BI型(SR-BI)和高密度脂蛋白的受体-配体复合体与胆固醇有关 通过反向胆固醇运输(RCT)从体内排出，在预防动脉粥样硬化方面至关重要。 我们研究的长期目标是了解调节SR-BI介导的机制 将胆固醇酯(CE)从高密度脂蛋白输送到肝脏排泄。这一应用的科学前提是 是基于越来越多的文献，这些文献表明，通过促进 通过增强胆固醇外流和RCT清除胆固醇。这一前提也得到突变的支持 在高密度脂蛋白胆固醇患者中发现的SCARB1(人类SR-BI基因)，阻止了选择性摄取 高密度脂蛋白胆固醇和增加心血管疾病的风险。虽然高密度脂蛋白-C的有效清除取决于RCT的最后几个步骤， 关于SR-BI/高密度脂蛋白相互作用触发高密度脂蛋白-CE选择性摄取的重要性的研究仍然有限。 在上一个资金周期中，我们最大的发现是第一个高分辨率的核磁共振结构 包含C-末端跨膜(TM)结构域并有助于SR-BI齐聚的SR-BI， 除了相邻的胞外区外，还含有一个具有独特疏水特性的短阿尔法螺旋。 在此应用程序中，我们基于这些令人兴奋的新发现和其他有希望的初步数据来测试 总体假设通过RCT的胆固醇流量是由SR-BI的结构特征驱动的，这些结构特征对 膜结合和受体齐聚。在目标1中，我们将使用结构导向突变和 一系列创新的生物物理技术，以确定受体/膜相互作用是否涉及 推测胞外膜旁螺旋是介导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到粪便的RCT来测试膜旁螺旋在体内的重要性。 在螺旋疏水性发生改变的地方表达突变的SR-BI受体。在目标2中，我们将使用尖端技术 核磁共振策略和顺磁松弛实验，以及结构引导突变，以确定 SR-BI寡聚体的组织，一种被认为是胆固醇从高密度脂蛋白中移动所必需的复合体 传给细胞。此外，我们将在SR-BI的TM域之间映射可能至关重要的精确绑定接口 在介导高密度脂蛋白-CE选择性摄取到质膜中。最后，在Aim 3中，使用非 SR-BI的寡聚突变体和相关对照，我们还将进行巨噬细胞到粪便的RCT研究 作为动脉粥样硬化的研究，确定SR-BI寡聚在小鼠模型中的体内功能相关性。 结合使用体外和体内研究，以及创新和最先进的技术，将 增进我们对SR-BI分子结构的了解。重要的是，我们的研究结果将 确定未来旨在预防高胆固醇血症及其相关病理的治疗方法，如 动脉粥样硬化，通过明确潜在的结构机制，推动SR-BI介导的选择性摄取 高密度脂蛋白-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.