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)利用长链脂肪酰基辅酶A和胆固醇两种亲脂性底物催化中性脂类胆固醇酯(CE)的形成。在单细胞水平上，ACAT通过将多余的胆固醇转化为CES来控制细胞膜胆固醇水平。在参与脂蛋白组装的细胞中，ACAT作为脂蛋白中中性脂核心的一部分提供CES。在病理生理条件下，ACAT参与在动脉粥样硬化斑块中形成富含CE的泡沫细胞。这项应用的等电点的长期目标是在分子水平上了解这种酶的工作原理。ACAT是一种微量存在于内质网中的膜结合酶。Pl的实验室通过功能互补鉴定了第一个ACAT基因(人ACAT1)。克隆的人ACAT1在CHO细胞和昆虫细胞中表达，经洗涤剂溶解后纯化为均一。目前，人ACAT1是膜结合型酰基转移酶超家族(至少由20个成员组成)中唯一被纯化为均一的成员。纯化的酶受胆固醇的变构控制。该酶是具有多个跨膜结构域的同源四聚体。在现有的各种分子试剂的情况下，在目前的提案中，我们要求资金来检验两个假设：a.ACAT1的催化可能在ER膜平面内完成。除了甾醇底物部位外，ACAT1还可能含有变构甾醇激活剂部位。我们有三个特定的目标：1.对与hACAT1固有相关的脂肪酰基-辅酶A水解酶活性进行生化表征。2.探索疏水性多肽(A.A.446-468)，包括推测的ACAT活性部位。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.