Molecular mechanism of a master regulator of sterol homeostasis

描述(由申请者提供)：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。SCAP整合到哺乳动物细胞的内质网(ER)膜上，结合胆固醇并经历配体门控的构象变化，调节SREBP转录因子的成熟。SCAP对细胞存活是必不可少的，介导他汀类药物降低低密度脂蛋白的活性，并可能作为心血管和代谢性疾病(如肝脏脂肪变性)的药物靶点。尽管SCAP具有生物学意义，但人们对其胆固醇结合和构象变化的生物物理机制知之甚少。SCAP的三维结构是什么？胆固醇结合如何改变SCAP与SREBP途径中下游蛋白(如COPII)的相互作用？为了了解胆固醇如何与SCAP结合，我们将设计一种适合生物物理特性和结构测定的SCAP胆固醇结合结构域(CBD)的可溶性形式。无胆固醇CBD结构和结合CBD结构的比较将为我们提供有关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突变和功能分析。为了发现SCAP如何将胆固醇结合与跨膜传播的更大规模的结构变化联系起来，并最终调节SREBP的运输，我们将确定一个生化稳定且易于处理的直系同系物，并通过单粒子低温EM和X射线结晶学解决整个跨膜区的结构。创新的蛋白质工程策略、低温电子显微镜和脂质介导的结晶学方法的结合将克服与膜蛋白相关的障碍，并提供这种生理必需的甾醇传感器的详细分子图像。在未来，我们将使用在这个项目中开发的知识和试剂来帮助发现小分子SCAP调节剂。

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.