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

鉴定促进 HDL-胆固醇清除的 SR-BI 结构特征

• 批准号: 10471386
• 负责人: Hayley R Powers
• 金额: $ 4.88万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471386
• 关键词: Antiatherogenic; Automobile Driving; Binding; Blood Circulation; C-terminal; Cardiovascular Diseases; Cause of Death; Cell membrane; Cells; Cholesterol; Cholesterol Esters; Cysteine; Data; Dependovirus; Dimerization; Electron Spin Resonance Spectroscopy; Electrons; Elements; Excision; Extracellular Domain; Feces; Future; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Homeostasis; Homology Modeling; Human; Hydrophobicity; Impairment; In Vitro; Knockout Mice; Knowledge; Leucine Zippers; Light; Lipids; Liver; Maps; Measurement; Measures; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutation; N-terminal; NMR Spectroscopy; Nuclear Magnetic Resonance; Outcome; Peptides; Physiological; Plasma; Process; Proteins; Relaxation; Research Design; Resolution; Role; SR-BI receptor; Site; Spin Labels; Structure; System; Techniques; Testing; Therapeutic; Translating; Transmembrane Domain; Tryptophan; Virus Diseases; Wild Type Mouse; alpha helix; cardiovascular disorder risk; cell type; clinically relevant; clinically significant; comparative; design; experimental study; extracellular; high density lipoprotein receptor; in vivo; in vivo Model; innovation; insight; macrophage; monomer; mutant; novel; particle; programs; protective effect; receptor; receptor function; reconstitution; reverse cholesterol transport; therapeutic development; tool; uptake

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.

项目摘要 高密度脂蛋白（HDL）是俗称为“好胆固醇”，由于其保护作用， 心血管疾病（CVD）。HDL被认为是抗动脉粥样硬化的，因为它能够从动脉中去除胆固醇。 胆固醇酯通过其受体清道夫受体BI（SR-BI）进入肝脏。的 HDL和SR-BI之间的相互作用是促进胆固醇净清除的最重要机制 因此，必须更好地了解促进身体健康的结构机制， HDL和SR-BI相互作用。在结构上，SR-BI由驱动受体功能的两个关键特征组成： 结合HDL和介导胆固醇递送所需的大细胞外结构域和（ii）两个锚定结构域 这些跨膜结构域与受体寡聚化有关。这项建议旨在 测试中心假设，即适当的SR-BI功能是由重要的SR-BI结构特征驱动的 用于膜结合和受体寡聚化。最近，我们的实验室成功地解决了高- SR-BI残基405-475的分辨率NMR结构，该肽是我们结构分析的最大工具。 问题研究该建议的第一个目的集中在SR-BI的细胞外元件，其有助于结合 和HDL-C的传递。SR-BI[405-475]肽包含C-末端跨膜结构域， 也是含有短螺旋的细胞外区域，在该提议中称为HSP 2。初步数据 提示HSP 2是脂质相关的，功能数据证明其在SR-BI介导的 胆固醇转运首先，我们将直接测量HSP 2内残基的质膜结合 使用创新的电子顺磁共振和色氨酸淬灭技术。然后，为了翻译 在体外观察到的功能变化与体内模型相比，破坏HSP 2疏水性的突变体将 引入SR-BI敲除小鼠。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，粪便逆转胆固醇转运。第二个目标处理的作用 跨膜结构域在SR-BI寡聚体形成中的作用，以及可能的胆固醇疏水通道 运动首先，将使用新的跨膜结构域定位C-末端跨膜结构域的二聚化界面。 顺磁弛豫增强方法。然后，我们计划解决一个高分辨率的结构的N- 通过核磁共振光谱法测定SR-BI的末端跨膜结构域。这些战略将使我们能够建立在 目前存在的结构信息，以形成一个更完整的故事SR-BI的寡聚状态。SR-BI是 对于维持脂质稳态具有重要的生理学意义，因为SR-BI突变的人表现出受损 胆固醇清除率，因此，心血管疾病的风险增加。阐明生产性SR-BI/HDL的机制 相互作用对于理解HDL-C清除和最终调节CVD风险至关重要。因此， 我们的研究结果可以将SR-BI确定为未来治疗的相关和有吸引力的靶点， 激活SR-BI介导的胆固醇转运并有效降低血浆胆固醇水平。