Molecular mechanism of a master regulator of sterol homeostasis

甾醇稳态主调节剂的分子机制

• 批准号: 9189632
• 负责人: Daniel Rosenbaum
• 金额: $ 30.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189632
• 关键词: Affinity; Behavior; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biophysical Process; Cardiovascular Diseases; Cardiovascular system; Cell Survival; Cells; Cholesterol; Cholesterol Homeostasis; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; Detergents; Disease; Drug Targeting; Electron Microscopy; Endoplasmic Reticulum; Engineering; Epitopes; Fatty Liver; Feedback; Foundations; Future; G-Protein-Coupled Receptors; Genetic Transcription; Goals; Health; Homeostasis; Human; Integral Membrane Protein; Knowledge; Length; Ligands; Link; Lipids; Low-Density Lipoproteins; Mammalian Cell; Mammals; Mediating; Membrane; Membrane Proteins; Metabolic; Metabolic Diseases; Methods; Micelles; Molecular; Molecular Conformation; Molecular Probes; Molecular Structure; Mutagenesis; Negative Staining; Orthologous Gene; Pathway interactions; Pharmaceutical Preparations; Physiological; Physiology; Positioning Attribute; Protein Engineering; Proteins; Public Health; Reagent; Recombinants; Resolution; Response Elements; Role; SCAP protein; Signal Transduction; Signaling Protein; Solubility; Sterols; Structural Models; Structure; System; Therapeutic; Transmembrane Domain; X-Ray Crystallography; base; biophysical properties; biophysical techniques; cholesterol control; improved; innovation; insight; lipid metabolism; mutant; overexpression; particle; polypeptide; protein E; protein structure; protein transport; public health relevance; sensor; small molecule; sterol homeostasis; therapeutic target; three dimensional structure; tool; transcription factor

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the molecular basis for the function of the cholesterol homeostasis signaling protein Scap. Integrated into the endoplasmic reticulum (ER) membrane of mammalian cells, Scap binds cholesterol and undergoes ligand-gated conformational changes that modulate the maturation of SREBP transcription factors. Scap is essential for cell survival, mediates the LDL-lowering activity of the statin drugs, and may be valuable as a drug target for cardiovascular and metabolic diseases such as hepatic steatosis. Despite the biological significance of Scap, remarkably little is known about the biophysical mechanism of its cholesterol binding and conformational changes. What is Scap's three- dimensional structure, and how is it changed by cholesterol binding to modulate Scap's interaction with downstream proteins(e.g. COPII) in the SREBP pathway? To understand how cholesterol binds to Scap, we will engineer a soluble form of Scap's cholesterol binding domain (CBD) that is amenable to biophysical characterization and structure determination. Comparison of cholesterol-free and bound CBD structures will provide our first molecular insights into sterol gating of Scap function, and will guide structure-based mutagenesis and functional assays. To discover how Scap links cholesterol binding to larger-scale structural changes that propagate across the membrane and ultimately modulate SREBP trafficking, we will identify an ortholog that is biochemically stable and tractable, and solve structures of the entire transmembrane region by single-particle cryoEM and X-ray crystallography. The combination of innovative protein engineering strategies, cryoEM, and lipid-mediated crystallography methods will overcome obstacles associated with membrane proteins and provide a detailed molecular picture of this physiologically essential sterol sensor. In the future we will use the knowledge and reagents developed in this project to help discover small-molecule Scap modulators.

描述（由申请人提供）：本项目的总体目标是了解胆固醇稳态信号蛋白Scap功能的分子基础。整合到哺乳动物细胞的内质网（ER）膜，Scap结合胆固醇，并经历配体门控构象变化，调节SREBP转录因子的成熟。Scap是细胞存活所必需的，介导他汀类药物的LDL降低活性，并且可能作为心血管和代谢性疾病如肝脂肪变性的药物靶点是有价值的。尽管Scap的生物学意义，显着很少有人知道它的胆固醇结合和构象变化的生物物理机制。Scap的三维结构是什么，它是如何通过胆固醇结合来调节Scap与SREBP途径中下游蛋白（例如COPII）的相互作用的？为了了解胆固醇如何与Scap结合，我们将设计一种可溶形式的Scap胆固醇结合结构域（CBD），该结构域适合生物物理表征和结构测定。无胆固醇和结合CBD结构的比较将提供我们对Scap功能的甾醇门控的第一个分子见解，并将指导基于结构的诱变和功能测定。为了发现Scap如何将胆固醇结合与跨膜传播并最终调节SREBP运输的大规模结构变化联系起来，我们将鉴定一种生物化学稳定且易于处理的直系同源物，并通过单颗粒cryoEM和X射线晶体学解决整个跨膜区域的结构。创新的蛋白质工程策略，cryoEM和脂质介导的晶体学方法的组合将克服与膜蛋白相关的障碍，并提供这种生理上必需的甾醇传感器的详细分子图像。在未来，我们将使用该项目中开发的知识和试剂来帮助发现小分子Scap调节剂。