FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

脂肪酸结合蛋白-配体特异性

• 批准号: 3242144
• 负责人: Friedhelm Schroeder
• 金额: $ 12.54万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-15 至 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3242144
• 关键词: Escherichia coli; L cell; acyl carrier protein; acyltransferase; chemical binding; cholesterol esters; enzyme mechanism; ergosterol; fatty acid binding protein; fluorescence spectrometry; fluorescent dye /probe; free fatty acids; gene expression; laboratory rat; lipid transport; protein purification; sterols; transfection; unsaturated fatty acids

The long range objective of the proposed research is to determine fundamental factors that regulate protein-lipid interactions within the cell. Specifically, the binding site(s) of fatty acids and sterols in fatty acid binding protein (FABP) [also called sterol carrier protein (SCP)] and the function of FABP in cholesterol esterification will be examined. This ubiquitous protein is present in microorganisms, plants, and animals, accounting for up to 14% of cytosolic protein. Despite this abundance little is know regarding 1) the function, 2) the binding specificities, affinities stoichiometries, and competition between exogenous ligands, and 3) the structures of the FABP/SCP proteins. A function in cellular fatty acid and/or sterol uptake and esterification has been proposed. Using fluorescent cholesterol analogues and time resolved fluorescence spectroscopy, we demonstrated for the first time that FABP/SCP binds sterols in vivo and in vitro. the approach is three-fold: 1) Isolate pure liver FABP/SCP and intestinal FABP/SCP which have two and one fatty acid binding sites, respectively. The coding region of the full length liver FABP and intestinal FABP cDNA will be expressed in E. coli and large quantities of the proteins will be isolated therefrom; 2) Use pure fluorescent sterols (cholestatrienol and dehydroergosterol), and fluorescent fatty acids (trans- and cis-parinaric acid) to characterize: a) the sterol binding site, b) the fatty acid binding site(s) and c) the competitive binding of sterols and fatty acids for the same or different binding sites on the FABP/SCP. since 60% of cytosolic free fatty acids are bound to FABP/SCP and FABP/SCP has similar Kd's for both fatty acids and fluorescent sterols, it seems highly likely that the cholesterol and the fatty acid carrying ability of FABP/SCP are mutually interdepending. 3) Determine the ability of these proteins to stimulate acyl-CoA Cholesteryl Acyl Transferase (ACAT) activity in vitro and in vivo under fatty acid free and fatty acid loaded conditions. The in vivo experiments will be performed with L cell fibroblasts transfected with the above protein(s). Results of these experiments should provide insights as to how a FABP/SCP bound lipid may modulate intracellular function. In addition, basic information regarding fatty acid interactions with a pure protein will be obtained.

拟议研究的长期目标是确定 调节蛋白质-脂质相互作用的基本因素 cell. 具体地，在一个或多个实施方案中，脂肪酸和固醇的结合位点在一个或多个实施方案中是不稳定的。 脂肪酸结合蛋白（FABP）[也称为固醇载体蛋白 （SCP）]和脂肪酸结合蛋白在胆固醇酯化中的作用 考察 这种无处不在的蛋白质存在于微生物，植物， 和动物，占细胞溶质蛋白的14%。 尽管如此 关于1）功能，2）绑定， 特异性、亲和性、化学计量学以及 外源配体，和3）FABP/SCP蛋白的结构。 一 在细胞脂肪酸和/或甾醇摄取和酯化中的功能 已经被提议了。 使用荧光胆固醇类似物和时间 解析荧光光谱，我们首次证明， FABP/SCP在体内和体外结合甾醇。 这一办法有三个方面： 1)分离出具有两种结构的纯肝FABP/SCP和肠FABP/SCP， 一个脂肪酸结合位点。 完整的编码区 将全长肝FABP和肠FABP cDNA在E.杆菌和 大量的蛋白质将从中分离; 2）使用纯的 荧光甾醇（胆甾三烯醇和脱氢麦角甾醇），和 荧光脂肪酸（反式和顺式-帕里那酸）来表征： a）甾醇结合位点，B）脂肪酸结合位点和c） 甾醇和脂肪酸的竞争性结合， FABP/SCP上的结合位点。 因为60%的胞质游离脂肪酸 与FABP/SCP和FABP/SCP结合的两种脂肪酸具有相似的Kd， 荧光固醇，这似乎很可能是胆固醇和 FABP/SCP的脂肪酸携带能力是相互依赖的。 第三章 测定这些蛋白质刺激酰基辅酶A胆固醇的能力 无脂肪酸条件下的体外和体内酰基转移酶（ACAT）活性 和脂肪酸负载条件。 体内实验将是 用上述蛋白质转染的L细胞成纤维细胞进行。 这些实验的结果应该提供关于FABP/SCP如何 结合的脂质可调节细胞内功能。 此外，基本 关于脂肪酸与纯蛋白质相互作用的信息将被 得到了