FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

拟议研究的长期目标是确定 调节体内蛋白质-脂质相互作用的基本因素 手机。具体来说，脂肪酸和甾醇的结合部位(S) 脂肪酸结合蛋白(FABP)[又称固醇载体蛋白 (SCP)]，FABP在胆固醇酯化中的作用将是 检查过了。这种普遍存在的蛋白质存在于微生物、植物、 和动物，占胞浆蛋白的14%。尽管如此 关于1)功能，2)绑定，我们知之甚少 特异性、亲和力、化学计量和相互竞争 外源配体，以及3)FABP/SCP蛋白的结构。一个 在细胞脂肪酸和/或甾醇摄取和酯化中的作用 已经被提出了。使用荧光胆固醇类似物和时间 分辨荧光光谱，我们第一次证明了 FABP/SCP在体内和体外都能与甾醇结合。这种方法有三个方面： 1)分离纯化的肝脏FABP/SCP和肠道FABP/SCP 一个脂肪酸结合部位分别为。全长基因的编码区 肝脏FABP和肠道FABP的长度将在大肠杆菌和 将从其中分离出大量的蛋白质；2)使用纯的 荧光甾醇(胆三烯醇和脱氢麦角甾醇)，以及 荧光脂肪酸(反式和顺式)表征： A)甾醇结合部位，b)脂肪酸结合部位(S)和c) 相同或不同的甾醇和脂肪酸的竞争性结合 FABP/SCP上的结合位点。因为60%的胞质游离脂肪酸是 与FABP/SCP和FABP/SCP结合的脂肪酸和 荧光甾醇，看起来很可能是胆固醇和 FABP/SCP的载脂能力是相互依赖的。3) 确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ACAT的体内外活性 和富含脂肪酸的条件下。体内实验将是 用转染上述蛋白的L细胞(S)进行细胞培养。 这些实验的结果应该提供关于FABP/SCP如何 结合脂可调节细胞内功能。此外，基本的 有关脂肪酸与纯蛋白质相互作用的信息将是 获得。

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.