FUNCTION ANALYSIS OF HUMAN ACAT

描述（由申请人提供）： 酰基辅酶A：胆固醇酰基转移酶（ACAT）利用两种亲脂底物，长链脂肪酰基辅酶A和胆固醇，催化形成中性脂质胆固醇酯（CE）。在单细胞水平，ACAT通过将过量胆固醇转化为CE来控制细胞膜胆固醇水平。在参与脂蛋白组装的细胞中，ACAT提供CE作为脂蛋白中中性脂质核心的一部分。在病理生理条件下，ACAT参与动脉粥样硬化斑块中富含CE的泡沫细胞的形成。本申请PI的长期目标是了解这种酶如何在分子水平上工作。ACAT是位于内质网中的微量膜结合酶。Pl的实验室通过功能互补鉴定了第一个ACAT基因（人ACAT 1）。在CHO细胞和昆虫细胞中表达的克隆的人ACAT 1已被洗涤剂溶解并纯化至均一。目前，人ACAT 1是膜结合酰基转移酶超家族（至少由20个成员组成）中唯一一个纯化至均一的成员。纯化的酶被证明是胆固醇的变构控制下。该酶是具有多个跨膜结构域的同源四聚体。随着各种分子试剂的出现，在目前的提案中，我们要求资金来验证两个假设：ACAT 1的催化作用可以在ER膜的平面内完成。B。ACAT 1除了固醇底物位点之外还可以含有变构固醇激活剂位点。我们有三个具体目标：1。生物化学表征与hACAT 1内在相关的脂肪酰辅酶A水解酶活性。2.为了探测疏水肽（a.a. 446-468），包含推定的ACAT活性位点。3.验证两个甾醇结合结构域假说。

DESCRIPTION (provided by applicant): Acyl coenzyme A:cholesterol acyltransferase (ACAT) utilizes two Iipophilic substrates, long-chain fatty acyl coenzyme A and cholesterol, to catalyze the formation of a neutral lipid cholesteryl ester (CE). At the single cell level, ACAT controls the cellular membrane cholesterol level by converting excess cholesterol into CEs. In cells involved in lipoprotein assembly, ACAT supplies CEs as part of the neutral lipid core in lipoproteins. Under pathophysiological conditions, ACAT is involved in forming CE-rich foam cells in the atherosclerotic plaques. The long-term goal of the PI of this application is to gain understanding of how this enzyme works at the molecular level. ACAT is a membrane bound enzyme located in the endoplasmic reticulum in minute quantity. The Pl's laboratory identified the first ACAT gene (human ACAT1) by functional complementation. The cloned human ACAT1 expressed in CHO cells and in insect cells has been solubilized by detergent and purified to homogeneity. At present, human ACAT1 is the only member of the membrane bound acyltransferase superfamily (comprised of at least 20 in numbers) that has been purified to homogeneity. The purified enzyme is shown to be under allosteric control by cholesterol. The enzyme is a homotetramer with multiple transmembrane domains. With various molecular reagents now available, in the current proposal, we request funds to test two hypotheses: A. Catalysis of ACAT1 may be completed within the plane of the ER membrane. B. ACAT1 may contain an allosteric sterol activator site in addition to a sterol substrate site. We enlist 3 Specific Aims: 1. To biochemically characterize the fatty acyI-CoA hydrolase activity intrinsically associated with hACAT1. 2. To probe the environment of the hydrophobic peptides (a.a. 446-468) comprising the putative ACAT active site. 3. To test the two sterol binding domain hypothesis.