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

通过 SR-BI 跨膜域的结构特征调节胆固醇转运

• 批准号: 9766411
• 负责人: Sarah C May
• 金额: $ 4.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2020-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9766411
• 关键词: Affinity; Arteries; Atherosclerosis; Bile fluid; Biliary; Binding; Biochemical; Blood Circulation; C-terminal; Cardiovascular Diseases; Cause of Death; Cells; Cholesterol; Cholesterol Esters; Complex; Data; Dimerization; Disease; Excision; Excretory function; Fellowship; Fluorescence Resonance Energy Transfer; Heart Diseases; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Hydrophobicity; Impairment; In Vitro; Inflammatory; Knowledge; Lead; Leucine Zippers; Liver; Maps; Mediating; Membrane; Molecular Conformation; Mutation; N-terminal; Pathway interactions; Peptides; Peripheral; Physiological; Plasma; Process; Proline; Regulation; Relaxation; Research; Resolution; Risk Factors; SR-BI receptor; Structure; Techniques; Testing; Therapeutic; Tissues; Transmembrane Domain; United States; cardioprotection; cardiovascular disorder risk; design; dimer; experimental study; flexibility; high density lipoprotein receptor; improved; in vivo; in vivo Model; monomer; mutant; novel therapeutic intervention; prevent; protein protein interaction; receptor; reverse cholesterol transport; three dimensional structure; uptake

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.

项目总结/摘要 心血管疾病（CVD）是美国的主要死亡原因。阿瑟费少校 心血管疾病的原因，是一种炎症性疾病，由于胆固醇积累在斑块沿着 动脉壁高密度脂蛋白（HDL）可以防止心血管疾病，因为它携带外周胆固醇到肝脏 在胆固醇逆向转运（RCT）过程中进行胆汁排泄。有效的RCT和选择性 从HDL摄取胆固醇酯（CE）进入细胞需要HDL与其高亲和力之间的相互作用 受体，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 促进HDL-CE的选择性摄取;然而，驱动寡聚化的机制仍然存在， 未知N-末端和C-末端跨膜结构域（分别为N-TMD和C-TMD）均为 SR-BI含有假定的二聚化基序，当破坏时，会损害SR-BI二聚化以及胆固醇 运输功能。最近，我们的实验室使用NMR技术解决了肽的高分辨率3D结构 的SR-BI（残基405-475），以及亮氨酸拉链二聚化基序。使用此 为了充分发挥我们的优势，我们在这个奖学金申请中设计了实验来验证假设 相邻SR-BI单体的跨膜结构域之间的SR-BI寡聚化和柔性 对于有效的RCT至关重要。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 SR-BI的胆固醇转运功能。在目标1.1中，我们将研究在细胞内脯氨酸扭结的意义。 C-TMD，而在目标1.2中，我们将检查SR-BI单体的C-TMD之间柔性降低的影响 使用“锁定二聚体”方法。在目标1.3中，我们将通过以下方式确定寡聚化的生理相关性： 在RCT的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 的SR-BI的N-TMD，以确定该域的结构特征，可能是功能相关的（目的 2.1）。然后，用两个跨膜结构域的NMR结构武装起来，我们将绘制二聚体界面， 使用顺磁弛豫支持C-TMD和/或N-TMD之间的SR-BI寡聚化 目标2.2中的增强战略。这些研究将有助于我们更好地了解SR-BI/HDL相互作用， 与SR-BI寡聚化一起，促进有效RCT并改善净胆固醇排泄。我们的研究结果 将有望设计出一类新的治疗方法， 胆固醇和预防动脉粥样硬化。