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。具体地说，我们建议检查结构和配体 重组脂肪酸中脂结合位点(S)的特异性 结合蛋白(FABP)[也称为甾醇载体蛋白(SCP)]， FABP/SCP在完整细胞脂肪酸摄取和固醇摄取中的作用 FABP/SCP参与大鼠的脂肪酸和甾醇代谢 在体外和在完整的细胞中。FABP/SCP是一种普遍存在的蛋白质 占细胞胞浆蛋白的14%。尽管一个巨大的 在过去20年中，对FABP/SCP的大量研究已经 为FABP/SCP在 细胞内脂肪酸的运输和利用，确凿证据 至于他们的生理机能(S)目前还没有。同样， 关于FABP/SCP配体结构的大部分信息 结合部位仅由氨基化合物的比较研究得出。 酸序列和二级结构预测。方法是 四个方面： 1)分离重组肝脏FABP/SCP，并将其分解为异构体 来自大肠杆菌的肠道FABP/SCP分别表达各自的cDNA。 体外或在L细胞成纤维细胞中磷酸化FABP/SCP 表达相应的cDNAs。 2)确定异构体和磷酸化在结构上的作用 FABP/SCP配体结合位点(S)，配体特异性(脂肪酸， 脂肪酰基COAs、胆固醇)和竞争性配体相互作用 放射性标记和荧光配基、光亲和标记、相 荧光法和圆二向色性 3)确定FABP/SCP改变脂肪代谢的能力 酸、脂酰化辅酶A和甾醇在体内外的表达 L细胞。 4.利用荧光和放射性标记脂肪酸检测 质膜脂肪酸转运系统及其与脂肪酸的相互作用 转导编码基因的L细胞胞浆FABP/SCP 肝脏或肠道FABP/SCP。 结构和分子生物学在这些方面的独特应用 实验旨在提供有关FABP/SCP如何绑定的新见解 配体可以调节细胞内的脂肪代谢，以及脂肪酸， 脂酰化辅酶A和甾醇与细胞内脂质转移相互作用 蛋白质。

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.