Role of ABCG5 and ABCG8 in Sterol Metabolism

胆固醇是一种刚性的疏水分子，在膜功能和胆汁酸及类固醇激素的合成中起重要作用。胆固醇在两种主要的人类疾病，冠心病和胆结石的发病机制中也起着核心作用。因此，维持胆固醇稳态的代谢途径已被广泛研究。2000年，Hobbs-Cohen实验室发现胆固醇排泄是由异二聚体ATP结合盒（ABC）转运蛋白介导的，该转运蛋白包括两个半转运蛋白ABCG 5（G5）和ABCG 8（G8）。破坏转运蛋白功能的突变会深刻地改变胆固醇代谢，并导致严重的早发冠心病。随后的研究表明，G5和G8在内质网中异二聚化，并运输到肠细胞和肝细胞的顶面，在那里它们将胆固醇和其他中性固醇转运到胆汁中。G5 G8的基因消融基本上消除了中性固醇的胆汁排泄，并且使用纯化的G5 G8蛋白的生化研究证实胆固醇是转运蛋白的直接底物。本项目的总体目标是明确G5 G8的生物学作用和功能机制。这笔赠款的资金支持了一项持续的研究工作，阐明了G5 G8在正常生理学中的作用，以及转运蛋白功能丧失和获得的临床后果和致病机制。本申请将集中在G5 G8的功能机制，以解决一个主要的未解决的问题，关于G5 G8的功能：G5 G8如何使用ATP水解的能量，跨生物膜转运刚性，疏水性，中性固醇分子？通常认为ABC转运蛋白利用ATP来驱动构象变化，从而导致底物跨膜转运。作为确定G5 G8的固醇转运的结构基础的第一步，X射线晶体学用于确定G5 G8在向内（IF）的开放构象中的结构，其先于ATP与转运蛋白的结合并允许底物的结合。为了确定驱动固醇流出的构象变化，现在将确定G5 G8在面向外的构象（OF）中的结构，其中底物被移动到细胞的外表面。在具体目标1中，将使用最先进的技术（X射线晶体学和cryo-EM）来获得G5 G8在运输循环的不同状态下的高分辨率结构。这些结构将用于生成和测试ATP水解如何导致底物转运的模型。具体目标2将检查甾醇结合位点和甾醇跨膜转运的沿着轨迹。为了实现这些目标，我们组建了一个多学科的研究团队，具有生理学，生物化学，合成化学和结构生物学方面的专业知识。

Cholesterol is a rigid, hydrophobic molecule that plays essential roles in membrane function and in the synthesis of bile acids and steroid hormones. Cholesterol also plays a central role in the pathogenesis of two major human diseases, coronary heart disease and gallstones. Accordingly, the metabolic pathways that maintain cholesterol homeostasis have been extensively studied. In 2000, the Hobbs-Cohen laboratory showed that cholesterol excretion is mediated by a heterodimeric ATP-Binding Cassette (ABC) transporter comprising two hemi-transporters ABCG5 (G5) and ABCG8 (G8). Mutations that disrupt the function of the transporter profoundly alter cholesterol metabolism and cause severe premature coronary heart disease. Subsequent studies showed that G5 and G8 heterodimerize in the endoplasmic reticulum and traffic to the apical surfaces of enterocytes and hepatocytes, where they transport cholesterol and other neutral sterols into bile. Genetic ablation of G5G8 essentially abolishes biliary excretion of neutral sterols, and biochemical studies using purified G5G8 protein confirmed that cholesterol is the direct substrate of the transporter. The overall goal of this Project is to define the biological role and functional mechanism of G5G8. Funding from this grant has supported a sustained research effort that has elucidated the role of G5G8 in normal physiology and the clinical consequences and pathogenic mechanism of both loss and gain of transporter function. This application will focus on the functional mechanism of G5G8 to address a major unresolved question regarding G5G8 function: How does G5G8 use the energy of ATP hydrolysis to translocate a rigid, hydrophobic, neutral sterol molecule across biological membranes? It is generally accepted that ABC transporters use ATP to power changes in conformation that result in translocation of substrate across the membrane. As a first step towards defining the structural basis of sterol transport by G5G8, X-ray crystallography was used to determine the structure of G5G8 in an inward-facing (IF), open conformation, which precedes binding of ATP to the transporter and allows for the binding of substrate. To define the conformational changes that drive sterol efflux the structures of G5G8 in the outward facing conformation (OF), in which substrate is moved to the exterior face of the cell, will now be determined. In Specific Aim 1, state-of-the-art technologies (both X-ray crystallography and cryo-EM) will be used to obtain high resolution structures of G5G8 in different states of the transport cycle. The structures will be used to generate and test models of how ATP hydrolysis leads to substrate transport. Specific Aim 2 will examine the sterol binding site and the trajectory along which sterols are translocated across the membrane. To accomplish these aims, we have assembled a multidisciplinary team of investigators with expertise in physiology, biochemistry, synthetic chemistry and structural biology.