FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

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. 具体来说，我们建议检查结构和配体 重组脂肪酸中脂质结合位点的特异性 结合蛋白（FABP）[也称为固醇载体蛋白（SCP）]， FABP/SCP在完整细胞中脂肪酸摄取和甾醇摄取中的作用， 以及FABP/SCP参与脂肪酸和甾醇代谢的作用。 体外和完整细胞中。 FABP/SCP是普遍存在的蛋白质 占细胞溶质蛋白的14%。 虽然一个巨大的 在过去的二十年里，对FABP/SCP的大量研究 为FABP/SCP的作用提供了大量间接证据， 细胞内脂肪酸转运和利用，确凿证据 对于它们的生理功能，目前还不清楚。 同样地， 关于FABP/SCP配体结构的大部分信息 结合位点仅来自于氨基的比较研究。 酸序列和二级结构预测。 该方法是 四重： 1)分离重组肝FABP/SCP并拆分为亚型， 来自表达相应cDNA的大肠杆菌的肠FABP/SCP。 在体外或L-细胞成纤维细胞中磷酸化FABP/SCP 表达各自的cDNA。 2)确定亚型和磷酸化对结构的作用 FABP/SCP配体结合位点，配体特异性（脂肪酸， 脂肪酰基辅酶A，胆固醇），以及与 放射性标记和荧光配体，光亲和标记，相位 荧光测定法和圆二色性 3)测定FABP/SCP改变脂肪代谢的能力 在体外和体内转染的细胞中， L细胞。 4.利用荧光和放射性标记的脂肪酸来检查 质膜脂肪酸转运系统及其与 在用编码FABP/SCP的cDNA转染的L细胞成纤维细胞中， 肝脏或肠FABP/SCP。 结构和分子生物学在这些领域的独特应用 实验的目的是提供新的见解，如何FABP/SCP结合 配体可以调节细胞内脂质代谢以及脂肪酸， 脂酰辅酶A和甾醇与细胞内脂质转移相互作用 proteins.

The long range objective of the proposed research is to determine fundamental factors that regulate protein-lipid interactions within the cell. Specifically, we propose to examine the structure and ligand specificity of the lipid binding site(s) in recombinant fatty acid binding protein (FABP) [also called sterol carrier protein (SCP)], the role of FABP/SCP in fatty acid uptake and sterol uptake in intact cells, and the participation of FABP/SCP in fatty acid and sterol metabolism in vitro and in intact cells. The FABP/SCPs are ubiquitous proteins representing up to 14% of cell cytosolic protein. Although a massive amount of research into the FABP/SCPs in the past two decades has provided much circumstantial evidence for a role of FABP/SCP in intracellular fatty acid transport and utilization, conclusive evidence for their physiological function(s) is not yet available. Likewise, most of the information regarding the structure of the FABP/SCP ligand binding site has only been derived from comparative studies of amino acid sequence and secondary structure predictions. The approach is four-fold: 1) Isolate and resolve into isoforms recombinant liver FABP/SCP and intestinal FABP/SCP from E coli expressing the respective cDNAs. Phosphorylate the FABP/SCP either in vitro or in L-cell fibroblasts expressing the respective cDNA. 2) Determine the role of isoforms and phosphorylation on the structure of the FABP/SCP ligand binding site(s), ligand specificity (fatty acids, fatty acyl CoAs, cholesterol), and competitive ligand interactions with radiolabeled and fluorescent ligands, photoaffinity labels, phase fluorometry, and circular dichroism 3) Determine the ability of FABP/SCPs to alter metabolism of fatty acids, fatty acyl CoAs, and sterol in vitro and in vivo in transfected L cells. 4. Utilize fluorescent and radiolabeled fatty acids to examine the plasma membrane fatty acid transport system and its interaction with cytosolic FABP/SCP in L cell fibroblasts transfected with cDNA encoding liver or intestinal FABP/SCP. The unique application of structural and molecular biology in these experiments is designed to provide new insights as to how FABP/SCP bound ligands may modulate intracellular lipid metabolism and how fatty acids, fatty acyl CoAs, and sterols interact with intracellular lipid transfer proteins.