Role of ABCG5 and ABCG8 in Sterol Metabolism

描述(申请人提供)：固醇代谢失调是两种主要人类疾病发展的关键因素：冠状动脉粥样硬化，西方社会的主要死亡原因；胆固醇结石，美国外科手术的主要指标之一。类固醇在体内运输的基本途径已经被很好地定义：类固醇通过内源性合成和肠道吸收进入体内，并通过肝脏以游离类固醇或转化为胆汁酸的形式排泄到胆汁中。参与这些过程的两个主要器官系统，肝脏和肠道，为类固醇在体内的积累提供了关键屏障，但这些屏障的分子组成和作用机制尚不清楚。一个有待解决的主要问题是，特定的细胞膜转运蛋白是否促进了胆固醇通过肠道和胆小管的转运。最近，对谷甾醇血症的研究提供了防止类固醇蓄积的分子机制的重要线索，谷甾醇血症是一种与类固醇吸收增加和类固醇排泄减少相关的遗传性疾病。最近，我们发现谷固醇血症是由于两种三磷酸腺苷结合盒(ABC)蛋白ABCG5和ABCG8的突变引起的。在谷固醇血症患者体内观察到的类固醇平衡的深刻变化表明，ABCG5和ABCG8可以限制肠道对类固醇的吸收，促进胆汁中类固醇的排泄，但对这两种蛋白的基本功能和特征几乎一无所知。这项拨款提案的总体目标是阐明ABCG5和ABCG8限制肠道中的类固醇吸收和促进肝细胞中的类固醇排泄的基本机制。建议的研究旨在解决三个关键问题：第一，ABCG5和ABCG8在细胞中的位置？第二，ABCG5和ABCG8的功能形式是什么？第三，ABCG5和ABCG8转运的底物(S)是什么？为了解决这些问题，我们提出了一系列全面的生化研究，以便对ABCG5/8运输系统进行直接和定量的调查。ABCG5和ABCG8的亚细胞定位将通过显微镜和细胞分级来确定。ABCG5和ABCG8的功能形式将通过免疫共沉淀研究来确定主要的二聚伙伴，然后通过纯化天然蛋白来确定运输复合体的其他成分。最后，将在蛋白脂质体中重组一个功能运输复合体，以确定转运蛋白的底物和基本的酶性质。这些研究将全面了解这一重要的运输系统预防高胆固醇血症和冠状动脉粥样硬化的分子机制。

DESCRIPTION (provided by applicant): The dysregulation of sterol metabolism is a critical factor in the development of two major human diseases: coronary atherosclerosis, the leading cause of death in the Western societies, and cholesterol gallstones, one of the leading indicators for surgery in the United States. The basic pathways of sterol transport in the body have been well defined: sterols enter the body via endogenous synthesis and intestinal absorption and are excreted via the liver into the bile, either as the free sterol or after conversion to bile acids. The two major organ systems involved in these processes, the liver and intestine, provide critical barriers to the accumulation of sterols in the body, but the molecular constituents and mechanisms of action of these barriers are poorly understood. A major unresolved issue that remains to be addressed is whether specific cell membrane transporters facilitate the translocation of cholesterol across the intestinal and bile canalicular membranes. An important clue to the molecular mechanisms that defend against sterol accumulation has recently emerged from studies of sitosterolemia, a genetic disorder associated with increased sterol absorption and decreased sterol excretion. Recently, we showed that sitosterolemia is due to mutations in two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8. The profound alteration in sterol homeostasis observed in sitosterolemic patients indicates that ABCG5 and ABCG8 serve to limit sterol absorption in the intestine and promote sterol excretion in the bile, but almost nothing is known about the basic function and characteristics of these two proteins. The overall goal of this grant proposal is to elucidate the basic mechanisms by which ABCG5 and ABCG8 limit sterol absorption in the intestine and facilitate sterol excretion in hepatocytes. The studies proposed are designed to address three critical questions: First, where are ABCG5 and ABCG8 located in the cell? Second, what is the functional form of ABCG5 and ABCG8? Third, what substrate(s) do ABCG5 and ABCG8 transport? To address these questions, we propose a comprehensive series of biochemical studies that will allow a direct and quantitative investigation of the ABCG5/8 transport system. The subcellular location of ABCG5 and ABCG8 will be determined by microscopy and cell fractionation. The functional form of ABCG5 and ABCG8 will be determined by co-immunoprecipitation studies to identify the primary dimerization partners, and then by purification of the native proteins to identify other components of the transport complex. Finally, a functional transport complex will be reconstituted in proteoliposomes to define the substrate and basic enzymatic properties of the transporter. These studies will provide a comprehensive understanding of the molecular mechanisms of this important transport system that protects against hypercholesterolemia and coronary atherosclerosis.