Role of ABCG5 and ABCG8 in Sterol Metabolism

ABCG5 和 ABCG8 在甾醇代谢中的作用

• 批准号: 7014053
• 负责人: Helen Haskell Hobbs
• 金额: $ 38.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014053
• 关键词: cell line; cell membrane; cholanate compound; dietary lipid; dimer; excretion; fluorescence microscopy; gastrointestinal absorption /transport; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; intestines; intracellular transport; laboratory mouse; laboratory rabbit; lipid metabolism; liposomes; liver cells; liver metabolism; membrane transport proteins; protein localization; protein purification; protein reconstitution; protein structure function; recombinant proteins; sterols; western blottings

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.

描述（由申请人提供）：固醇代谢的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）和胆固醇结石（美国外科手术的主要指标之一）。固醇在体内转运的基本途径已经得到很好的定义：固醇通过内源性合成和肠道吸收进入体内，并通过肝脏排泄到胆汁中，作为游离固醇或转化为胆汁酸。参与这些过程的两个主要器官系统，肝脏和肠道，为体内固醇的积累提供了关键屏障，但对这些屏障的分子组成和作用机制知之甚少。一个主要的悬而未决的问题，仍然有待解决的是是否特定的细胞膜转运蛋白促进胆固醇跨肠和胆管膜易位。一个重要的线索，防止固醇积累的分子机制，最近出现的研究谷甾醇血症，一种遗传性疾病与增加固醇吸收和减少固醇排泄。最近，我们发现谷甾醇血症是由于两个ATP结合盒（ABC）蛋白ABCG 5和ABCG 8的突变。在谷固醇血症患者中观察到的固醇稳态的深刻改变表明ABCG 5和ABCG 8用于限制肠中的固醇吸收并促进胆汁中的固醇排泄，但几乎对这两种蛋白质的基本功能和特征一无所知。这项资助提案的总体目标是阐明ABCG 5和ABCG 8限制肠道中固醇吸收并促进肝细胞中固醇排泄的基本机制。这些研究旨在解决三个关键问题：第一，ABCG 5和ABCG 8在细胞中的位置？第二，ABCG 5和ABCG 8的函数形式是什么？第三，ABCG 5和ABCG 8转运什么底物？为了解决这些问题，我们提出了一个全面的一系列的生化研究，将允许直接和定量调查的ABCG 5/8运输系统。ABCG 5和ABCG 8的亚细胞位置将通过显微镜和细胞分级分离来确定。ABCG 5和ABCG 8的功能形式将通过共免疫沉淀研究来确定，以鉴定初级二聚化伴侣，然后通过纯化天然蛋白质来鉴定转运复合物的其他组分。最后，功能性转运复合物将在蛋白脂质体中重构，以确定转运蛋白的底物和基本酶性质。这些研究将提供一个全面的了解这一重要的运输系统，防止高胆固醇血症和冠状动脉粥样硬化的分子机制。