Role of ABCG5 and ABCG8 in Sterol Metabolism

ABCG5 和 ABCG8 在甾醇代谢中的作用

• 批准号: 8029566
• 负责人: Helen Haskell Hobbs
• 金额: $ 43.84万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8029566
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adrenal Glands; Affect; Amino Acid Substitution; Animals; Bile Acids; Bile fluid; Biliary; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cause of Death; Cells; Cholelithiasis; Cholesterol; Cholesterol Homeostasis; Collaborations; Coronary; Coronary Arteriosclerosis; Couples; Coupling; Dietary Sterol; Disease; Endoplasmic Reticulum; Enterocytes; Excretory function; Foundations; Functional disorder; Funding; Goals; Grant; Health; Hepatic Tissue; Hepatocyte; Human; Hydrolysis; In Vitro; Intestinal Absorption; Intestines; Liver; Maintenance; Mediating; Membrane; Membrane Lipids; Metabolism; Modeling; Molecular; Molecular Genetics; Mus; Mutation; Nucleotides; Operative Surgical Procedures; Pathway interactions; Physiological; Physiology; Phytosterols; Plants; Play; Process; Proteins; Reagent; Recombinant Proteins; Recombinants; Relative (related person); Research; Research Infrastructure; Research Personnel; Role; Series; Sitosterol; Sitosterols; Societies; Sterols; Substrate Specificity; System; Testing; Tissues; United States; Vertebrates; absorption; apical membrane; base; design; human disease; in vitro Assay; in vivo; mutant; overexpression; palmitoylation; polarized cell; premature; prevent; programs; reconstitution; research study; reverse cholesterol transport; tool

DESCRIPTION (provided by applicant): The dysregulation of sterol metabolism contributes to two major human diseases: coronary atherosclerosis, a major cause of death in Western societies, and cholesterol gallstones, a leading indicator 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 molecular mechanism responsible for the secretion of cholesterol by the liver and intestine was revealed by the finding that mutations in either of two ATP binding cassette (ABC) half-transporters, ABCG5 (G5) and ABCG8 (G8) cause sitosterolemia, an autosomal recessive disease characterized by accumulation of cholesterol and plant-derived sterols, and premature coronary atherosclerosis. This discovery was the starting point of this grant. During the last funding period, we elucidated the basic biology and pathophysiology of G5G8: we found that the two proteins form a heterodimer in the endoplasmic reticulum (ER) that is transported to the apical membranes of hepatocytes or enterocytes, and that most mutations causing sitosterolemia interfere with the coupling and transport of G5G8. We developed mice in which G5 and G8 were deleted, or overexpressed, and provided direct evidence that G5G8 prevents the accumulation of cholesterol and non-cholesterol sterols, and are essential for the maintenance of cholesterol homeostasis in the liver and in extra-hepatic tissues such as the adrenal. A major unresolved question arising from these studies is the role of G5G8 in the intestine. It is generally believed that biliary excretion is the major pathway for cholesterol excretion in humans, but we, and others, have found that fecal sterol excretion is normal in mice unable to secrete cholesterol into bile. This finding suggests that the intestine may play a major role in the elimination of cholesterol. Here we have designed a series of studies to address specifically the role of G5G8 in the intestine in protecting against accumulation of dietary sterols and in promoting the reverse cholesterol transport of endogenously-derived sterols. We have also developed the first in vitro assays for sterol transport by G5G8. We have purified and reconstituted the native (and recombinant) proteins, and shown that sterols are direct transport substrates of the heterodimer. We propose now to use these assays to address fundamental questions regarding the mechanism by which G5G8 couples ATP hydrolysis to the translocation of sterols across membranes. How does G5G8 recognize sterols and make them available to acceptors in the bile and intestinal lumen? We have the expertise, molecular tools and infrastructure to address these important physiological and biochemical questions.

描述（由申请人提供）：固醇代谢的失调导致两种主要的人类疾病：冠状动脉粥样硬化，这是西方社会的主要死亡原因，胆固醇结石，这是美国外科手术的主要指标。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。通过以下发现揭示了负责肝脏和肠道分泌胆固醇的分子机制：两种ATP结合盒（ABC）半转运蛋白ABCG 5（G5）和ABCG 8（G8）中的任一种突变导致谷甾醇血症，这是一种常染色体隐性疾病，其特征在于胆固醇和植物来源的甾醇的积累和过早的冠状动脉粥样硬化。这一发现是这项资助的起点。在上一个资助期间，我们阐明了G5G8的基础生物学和病理生理学：我们发现这两种蛋白质在内质网（ER）中形成异二聚体，转运到肝细胞或肠上皮细胞的顶膜，并且大多数导致谷甾醇血症的突变都会干扰G5G8的偶联和转运。我们开发了G5和G8缺失或过表达的小鼠，并提供了直接证据表明G5G8可防止胆固醇和非胆固醇的积累，并且对于维持肝脏和肝外组织（如肾上腺）中的胆固醇稳态至关重要。从这些研究中产生的一个主要未解决的问题是G5G8在肠道中的作用。一般认为，胆汁排泄是人体胆固醇排泄的主要途径，但我们和其他人发现，不能将胆固醇分泌到胆汁中的小鼠的粪便固醇排泄是正常的。这一发现表明，肠道可能在胆固醇的消除中发挥重要作用。在这里，我们设计了一系列的研究，以解决具体的作用，G5G8在肠道中防止积累的膳食固醇和促进胆固醇的内源性衍生的固醇的反向运输。我们还开发了第一个G5 G8固醇转运的体外试验。我们已经纯化和重组的天然（和重组）蛋白质，并表明，甾醇是异二聚体的直接运输底物。我们现在建议使用这些检测来解决有关G5G8将ATP水解与甾醇跨膜转运偶联的机制的基本问题。G5G8如何识别固醇并使其可用于胆汁和肠腔中的受体？我们拥有解决这些重要生理和生化问题的专业知识、分子工具和基础设施。