FUNCTION ANALYSIS OF HUMAN ACAT

人类ACAT的功能分析

• 批准号: 6873019
• 负责人: Ta Yuan CHANG
• 金额: $ 35.55万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873019
• 关键词: active sites; acyl coA; acyltransferase; affinity labeling; allosteric site; biochemistry; biophysics; catalyst; cholesterol; endoplasmic reticulum; enzyme activity; enzyme structure; lipid bilayer membrane; protein binding; site directed mutagenesis; sterols

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.

描述（由申请人提供）： 酰基辅酶A：胆固醇酰基转移酶（ACAT）利用两种亲脂性底物，长链脂肪脂肪酰基辅酶A和胆固醇，以催化中性脂质胆固醇的形成。在单细胞水平上，ACAT通过将多余的胆固醇转化为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的一部分。在病理生理条件下，ACAT参与了动脉粥样硬化斑块中形成富含CE的泡沫细胞。本应用PI的长期目标是了解该酶在分子水平上的工作方式。 ACAT是一种膜结合的酶，位于内质网中的微量含量。 PL的实验室通过功能补充确定了第一个ACAT基因（人ACAT1）。在CHO细胞和昆虫细胞中表达的克隆的人ACAT1已通过洗涤剂溶解并纯化为均匀性。目前，人ACAT1是膜结合的酰基转移酶超家族的唯一成员（由至少20个）纯化为同质性。纯化的酶显示在胆固醇的变构控制下。该酶是具有多个跨膜结构域的同二探测器。现在，在当前的提案中，我们要求资金来检验两个假设：A。A. ACAT1的催化可以在ER膜的平面内完成。 B. ACAT1除了固醇底物位点外，还可以包含变构固醇活化剂位点。我们提出了3个特定目标：1。生化表征与HACAT1本质上相关的脂肪ACYI-COA水解酶活性。 2。探测疏水肽的环境（A.A. 446-468），其中包括推定的ACAT活性位点。 3。测试两个固醇结合结构域假设。