Identification of structural features of SR-BI that facilitate HDL-cholesterol clearance

项目总结 高密度脂蛋白(Hdl)被俗称为“好胆固醇”，因为它有预防胆固醇的作用。 心血管疾病(CVD)。高密度脂蛋白被认为是抗动脉粥样硬化的，因为它能从 并通过其受体清道夫受体BI(SR-BI)将胆固醇酯输送到肝脏。这个 高密度脂蛋白和SR-BI之间的相互作用是促进胆固醇净去除的最重要机制 因此，必须更好地理解促进 高密度脂蛋白和SR-BI的相互作用。从结构上讲，SR-BI由驱动受体功能的两个关键特征组成：(I)a 结合高密度脂蛋白和介导胆固醇转运所需的大胞外域和(Ii)两个锚定 与受体齐聚有关的跨膜结构域。这项提议旨在 检验以下中心假设：正确的SR-BI功能是由SR-BI的重要结构特征驱动的 用于膜结合和受体齐聚。最近，我们的实验室成功地解决了高- 解析SR-BI残基405-475的核磁共振结构，这是我们研究结构的最大工具 学习。这项建议的第一个目标集中在SR-BI的细胞外元件上，这些元件有助于结合 和高密度脂蛋白-C的传递。SR-BI[405-475]肽包含C-末端跨膜结构域和 也是包含短螺旋的胞外区，在本提案中称为螺旋2。初步数据 提示Helix 2与脂质相关，功能数据表明其在SR-BI介导中的重要性 胆固醇的运输。首先，我们将直接测量Helix 2内残基的质膜结合 使用创新的电子顺磁共振和色氨酸猝灭技术。然后，为了将 在体外观察到体内模型的功能变化，破坏Helix 2疏水性的突变体将 被引入SR-BI基因敲除小鼠。然后我们将测量这些突变体对巨噬细胞的影响- 与野生型小鼠相比，粪便可以逆转胆固醇的运输。第二个目标是解决 SR-BI低聚物形成中的跨膜结构域，可能是胆固醇的疏水隧道 有动静。首先，C-末端跨膜结构域的二聚化界面将使用新的 顺磁驰豫增强方法。然后我们计划解析N-的高分辨率结构- 核磁共振波谱分析SR-BI的末端跨膜结构域。这些战略将使我们能够在 目前存在的结构信息，以形成一个更完整的故事的SR-BI的低聚状态。SR-BI是 由于SR-BI突变的人表现出受损，因此对维持脂质平衡具有重要的生理学意义 胆固醇清除，因此，心血管疾病的风险增加。阐明产生SR-BI/HDL码的机制 相互作用对于了解高密度脂蛋白胆固醇清除和最终调节心血管疾病风险至关重要。因此， 我们的研究结果可以确定SR-BI是未来治疗的相关和有吸引力的靶点，旨在 激活SR-BI介导的胆固醇转运，有效降低血浆胆固醇水平。

PROJECT SUMMARY High density lipoprotein (HDL) is colloquially known as ‘good cholesterol’ due to its protective effects against cardiovascular disease (CVD). HDL is considered anti-atherogenic due to its ability to remove cholesterol from the periphery and deliver cholesteryl ester to the liver via its receptor, scavenger receptor BI (SR-BI). The interaction between HDL and SR-BI is the most important mechanism that facilitates net removal of cholesterol from the body, and as such, it is imperative to better understand the structural mechanisms that promote the HDL and SR-BI interaction. Structurally, SR-BI consists of two key features that drive receptor function: (i) a large extracellular domain required to bind HDL and mediate cholesterol delivery and (ii) two anchoring transmembrane domains which have been implicated in receptor oligomerization. This proposal is designed to test the central hypothesis that proper SR-BI function is driven by structural features of SR-BI that are important for membrane association and receptor oligomerization. Recently, our lab was successful in solving the high- resolution NMR structure of SR-BI residues 405-475 and this peptide serves as our biggest tool in structural studies. The first Aim of this proposal focuses on the extracellular elements of SR-BI that contribute to binding and delivery of HDL-C. The SR-BI[405-475] peptide encompasses the C-terminal transmembrane domain and also an extracellular region containing a short  helix, referred to in this proposal as Helix 2. Preliminary data suggests Helix 2 is lipid-associated and functional data demonstrate its importance in SR-BI-mediated cholesterol transport. First, we will directly measure plasma membrane association of residues within Helix 2 using innovative electron paramagnetic resonance and tryptophan quenching techniques. Then, to translate the observed in vitro functional changes to an in vivo model, mutants that disrupt the hydrophobicity of Helix 2 will be introduced into SR-BI knockout mice. We will then measure the effect these mutants have on macrophage- to-feces reverse cholesterol transport compared to wildtype mice. The second Aim tackles the role of the transmembrane domains in the formation of SR-BI oligomers and possibly, a hydrophobic tunnel for cholesterol movement. First, the dimerization interface of the C-terminal transmembrane domain will be mapped using novel paramagnetic relaxation enhancement methods. We then plan to resolve a high-resolution structure of the N- terminal transmembrane domain of SR-BI by NMR spectroscopy. These strategies will allow us to build upon currently-existing structural information to form a more complete story of SR-BI’s oligomeric state. SR-BI is physiologically important for maintaining lipid homeostasis, as humans with mutations in SR-BI display impaired cholesterol clearance and, hence, an elevated risk of CVD. Clarifying the mechanisms of productive SR-BI/HDL interactions is vital to understanding HDL-C clearance and ultimately modulating CVD risk. As such, the outcomes of our studies could identify SR-BI as a relevant and attractive target for future therapeutics aimed at activating SR-BI-mediated cholesterol transport and effectively lowering plasma cholesterol levels.