STRUCTURE AND FUNCTION OF MEMBRANE TRANSPORT PROTEINS

膜运输蛋白的结构和功能

• 批准号: 2377732
• 负责人: DANIEL S LEVY
• 金额: $ 25.04万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-08-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2377732
• 关键词: MDCK cell; active sites; basolateral membrane; biological transport; cholates; confocal scanning microscopy; endoplasmic reticulum; epoxide hydrolase; fluorescence microscopy; intracellular transport; laboratory rat; liver cells; liver metabolism; membrane transport proteins; protein structure function; receptor binding; site directed mutagenesis; transfection; vesicle /vacuole

Bile acids such as taurocholic acid are organic anions that play a critical role in numerous physiological processes such as a) digestion, b) formation of bile, which functions as an secretory vehicle for numerous compounds such as cholesterol and metabolites of exogenous drugs and carcinogens, and c) regulation of cholesterol homeostasis by modulating its synthesis and degradation. Defects in the transport of bile acids result in intrahepatic cholestasis and numerous pathological conditions including liver injury and failure. A sinusoidal plasma membrane protein mediating sodium-dependent taurocholate uptake by hepatocytes has been isolated and shown to be indistinguishable from microsomal epoxide hydrolase (mEH). This protein, which also mediates the transport of taurocholate into vesicles derived from the smooth endoplasmic reticulum has been shown to exist in two distinct topological orientations in this cell compartment, one of which is targeted to the plasma membrane. The long term goals of this project are to investigate the molecular mechanism and regulation of this important liver transport system as well as delineating its physiological role in the uptake of bile acids and related substrates by a) characterizing the architecture, substrate binding site and functional properties of this transport system in the plasma membrane and endoplasmic reticulum; b) defining the mechanism which regulates the generation of multiple topologies and targeting of this single protein and c) elucidating the mechanism of transcellular movement of bile acids from the blood to the bile compartment. The specific aims of this proposal are: 1) to characterize architectural and functional aspects of this bile acid transport (BAT) protein as expressed in hepatocyte sinusoidal plasma membranes and smooth endoplasmic reticulum membranes using a) sequence- specific anti-peptide antibodies (SSAPA) in conjunction with confocal fluorescence microscopy and immunogold electron microscopy, and b) site- specific and membrane domain-specific chemical and enzymatic reagents; 2) to characterize the topology, transport function and substrate specificity of the BAT protein expressed in the basolateral plasma membrane and endoplasmic reticulum of MDCK cells, and the role of intracellular vesicles in the transcellular movement of bile acids, using cDNA transfection procedures in conjunction with SSAPA and confocal microscopy and 3) to evaluate the role of specific amino acid residues in a) determining the membrane topology and cell targeting of BAT and b) the mechanism of a bile acid transport, using site-directed mutagenesis and transfection procedures. These studies will provide a detailed understanding of the structure, mechanism of action and physiological role of this important transport system, as well as elucidating factors involved in regulating transport characteristics, membrane protein topological orientation and targeting.

胆汁酸如牛磺胆酸是有机阴离子， 在许多生理过程中起关键作用，例如a）消化，B） 胆汁的形成，其作为许多分泌媒介物起作用。 化合物如胆固醇和外源性药物的代谢物， 致癌物，和c）通过调节胆固醇稳态来调节胆固醇稳态， 其合成和降解。胆汁酸转运缺陷 导致肝内胆汁淤积和许多病理状况 包括肝损伤和衰竭一种窦状质膜蛋白 肝细胞介导钠依赖性牛磺胆酸盐摄取， 分离并显示与微粒体环氧化物无法区分 水解酶（mEH）。这种蛋白质，也介导运输 牛磺胆酸钠进入来自滑面内质网的囊泡 已经被证明存在于两个不同的拓扑方向，在这个 细胞隔室，其中之一是针对质膜。的 本项目的长期目标是研究分子机制 和调节这一重要的肝脏运输系统， 描述其在胆汁酸摄取和相关的生理作用 a）表征结构，底物结合位点 以及质膜中该转运系统的功能特性 和内质网; B）定义调节内质网的机制， 产生多个拓扑结构并靶向该单一蛋白质， c）阐明胆汁酸的跨细胞移动的机制， 血液进入胆汁室这项建议的具体目标是： 1)为了表征这种胆汁酸的结构和功能方面， 肝细胞窦状隙血浆中表达的转运（BAT）蛋白 膜和平滑内质网膜，使用a）序列- 特异性抗肽抗体（SSAPA）结合共聚焦显微镜 荧光显微术和免疫金电子显微术，和B）位点- 特异性和膜结构域特异性化学和酶试剂; 2） 以表征拓扑结构、转运功能和底物特异性 BAT蛋白在基底外侧质膜中表达， MDCK细胞的内质网，以及细胞内的作用 胆汁酸跨细胞运动中的囊泡，使用cDNA 转染程序结合SSAPA和共聚焦显微镜 和3）评估特定氨基酸残基在a）中的作用 确定BAT的膜拓扑结构和细胞靶向，和B） 胆汁酸转运的机制，使用定点诱变， 转染程序。这些研究将提供详细的 了解结构、作用机制和生理作用 这一重要的运输系统，以及阐明因素 参与调节转运特性，膜蛋白 拓扑定向和目标定位。