Regulation of cholesterol transport by structural features in SR-BI’s transmembrane domains

项目摘要/摘要 心血管疾病(CVD)是美国的主要死亡原因。动脉粥样硬化，主要 心血管疾病的病因，是一种炎症性疾病，由胆固醇在斑块中沿 动脉壁。高密度脂蛋白(高密度脂蛋白)可以预防心血管疾病，因为它将外周胆固醇输送到肝脏。 在被称为反向胆固醇转运(RCT)的过程中用于胆汁排泄。有效的RCT和选择性 从高密度脂蛋白中摄取胆固醇酯(CE)进入细胞需要高密度脂蛋白及其高亲和力之间的相互作用 清道夫受体-BI(SR-BI)。SR-BI二聚体和高阶低聚物的存在是重要的 为了促进对高密度脂蛋白-CE的选择性摄取；然而，驱动齐聚的机制仍然存在 未知。N-末端和C-末端跨膜结构域(分别为N-TMD和C-TMD) SR-BI含有假定的二聚化基序，当被破坏时，会削弱SR-BI的二聚化以及胆固醇 运输功能。最近，我们实验室使用了核磁共振技术来解析多肽的高分辨率三维结构 跨越C-TMD的SR-BI(残基405-475)，以及亮氨酸拉链二聚基序。使用这个 结构，我们在这个奖学金应用程序中设计了实验来检验这一假设 SR-BI齐聚和相邻SR-BI单体跨膜区之间的柔韧性 对于有效的随机对照试验是必不可少的。在目标1中，我们将测试SR-BI的C-TMD的关键结构特征的重要性 关于SR-BI的胆固醇转运功能。在目标1.1中，我们将研究 C-TMD，而在Aim 1.2中，我们将研究SR-BI单体的C-TMD之间灵活性降低的影响 使用“锁定调光器”的方法。在目标1.3中，我们将通过以下方式确定齐聚的生理学相关性 在RCT体内模型中测试亮氨酸拉链SR-BI突变体。在目标2中，我们将确定核磁共振结构 SR-BI的N-TMD，以便识别可能在功能上相关的该域的结构特征(AIM 2.1)。然后，有了这两个跨膜结构域的核磁共振结构，我们将映射二聚体界面(S)， 顺磁驰豫支持C-TMDs和/或N-TMDs之间的SR-BI齐聚 目标2.2中的增强策略。这些研究将帮助我们更好地理解SR-BI/HDL是如何相互作用的， 与SR-BI齐聚一起，促进有效的RCT并改善净胆固醇排泄。我们的发现 将有望导致设计一种新的治疗方法，旨在改善血浆的清除 胆固醇和预防动脉粥样硬化。

PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in the United States. Atherosclerosis, the major cause of CVD, is an inflammatory disease resulting from the accumulation of cholesterol in plaque along the artery walls. High density lipoprotein (HDL) protects against CVD, as it carries peripheral cholesterol to the liver for biliary excretion in a process known as reverse cholesterol transport (RCT). Efficient RCT and the selective uptake of cholesteryl esters (CE) from HDL into cells require interactions between HDL and its high affinity receptor, scavenger receptor-BI (SR-BI). The presence of SR-BI dimers and higher-order oligomers is important for facilitating the selective uptake of HDL-CE; however, the mechanisms that drive oligomerization remain unknown. Both the N-terminal and C-terminal transmembrane domains (N-TMD and C-TMD, respectively) of SR-BI contain putative dimerization motifs that, when disrupted, impair SR-BI dimerization as well as cholesterol transport functions. Recently, our lab used NMR techniques to solve the high-resolution 3D structure of a peptide of SR-BI (residues 405-475) that spans the C-TMD, as well as a leucine zipper dimerization motif. Using this structure to our full advantage, we have designed experiments in this fellowship application to test the hypothesis that SR-BI oligomerization and flexibility between the transmembrane domains of neighboring SR-BI monomers are essential for efficient RCT. In Aim 1, we will test the importance of key structural features of SR-BI’s C-TMD on cholesterol transport functions of SR-BI. In Aim 1.1, we will study the significance of a proline kink within the C-TMD, while in Aim 1.2, we will examine the effects of reduced flexibility between C-TMDs of SR-BI monomers using a “locked dimer” approach. In Aim 1.3, we will determine the physiological relevance of oligomerization by testing the leucine zipper SR-BI mutant in an in vivo model of RCT. In Aim 2, we will determine the NMR structure of SR-BI’s N-TMD in order to identify structural features of this domain that may be functionally relevant (Aim 2.1). Then, armed with NMR structures of both transmembrane domains, we will map the dimer interface(s) that support SR-BI oligomerization between the C-TMDs and/or N-TMDs using paramagnetic relaxation enhancement strategies in Aim 2.2. These studies will help us to better understand how SR-BI/HDL interactions, together with SR-BI oligomerization, promote efficient RCT and improve net cholesterol excretion. Our findings will hopefully lead to the design of a new class of therapeutics directed at improving clearance of plasma cholesterol and preventing atherosclerosis.