Functional Analysis of ACAT

描述(申请人提供)：这项研究的长期目标是了解酰基辅酶A：胆固醇酰基转移酶(ACAT)在人类健康和疾病中的作用，特别是在动脉粥样硬化方面，动脉粥样硬化是全球发病率和死亡率的主要原因。目前的建议有两个主要目标：继续进行ACAT1的体外结构-功能分析，并在体内检测巨噬细胞ACAT1在APOE-/-小鼠模型和Ldlr-/-apoB48缺陷小鼠动脉粥样硬化模型中的作用。作为细胞胆固醇代谢的关键酶，ACAT利用胆固醇和长链脂肪酰基辅酶A来产生胆固醇酯，并被自己的底物胆固醇变构激活，以防止内质网胆固醇的过度积聚。ACAT1是巨噬细胞中ACAT的主要同工酶，在动脉粥样硬化早期泡沫细胞的形成中起关键作用。ACAT1在许多其他类型的细胞中也有表达，包括骨髓中的造血干细胞。ACAT1在不同组织中的生理作用仍有待仔细研究。因此，该项目的目标是：1)确定参与ACAT1底物结合、催化和/或变构控制的关键氨基酸残基；2)描述失活巨噬细胞Acat1在动脉粥样硬化发生和发展阶段的作用。我们将使用一种新的重组hACAT1酶的表达和纯化方法和七种不同的生化分析来实现目标1，并将使用新开发的巨噬细胞特异性Acat1 KO小鼠模型(Acat1-M/-M)作为新的试剂来实现目标2。ACAT1是治疗人类阿尔茨海默病和其他疾病的潜在靶点。目前可用的大多数ACAT抑制剂都会引起非特异性的细胞毒性，并抑制具有类似活性部位的其他酶，可能会导致其他不良副作用。这一应用的结果将帮助研究人员设计副作用最小的ACAT1特异性抑制剂，并有助于确定ACAT1作为治疗动脉粥样硬化的靶点的实用性。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the role of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in human health and disease, particularly in atherosclerosis, a leading cause of morbidity and mortality worldwide. The current proposal has two main goals: to continue to conduct structure-function analysis of ACAT1 in vitro, and to examine the role of macrophage ACAT1 in vivo in the Apoe-/- mouse model and the Ldlr-/- apoB48-deficient mouse model for atherosclerosis. As a key enzyme in cellular cholesterol metabolism, ACAT utilizes cholesterol and long-chain fatty acyl coenzyme A to produce cholesteryl esters, and is allosterically activated by its own substrate, cholesterol, to guard against excess buildup of cholesterol at the endoplasmic reticulum. ACAT1 is the major isoenzyme of ACAT in macrophages and plays a key role in foam cell formation in the early stages of atherosclerosis. ACAT1 is also expressed in many other cell types, including hematopoietic stem cells in the bone marrow. The physiological roles of ACAT1 in various tissues remain to be carefully investigated. Thus, the aims of this project are 1) to identify key amino acid residues involved in substrate binding, catalysis, and/or allosteric control of ACAT1; and 2) to delineate the effects of inactivating macrophage Acat1 during the initiation and progression stages of atherosclerosis. We will employ a new procedure for expression and purification of the recombinant hACAT1 enzyme and seven different biochemical assays to pursue Aim 1, and will employ a newly developed macrophage-specific Acat1 KO mouse (Acat1-M/-M) model as a novel reagent to pursue Aim 2. ACAT1 is a potential target for treating Alzheimer disease and other diseases in humans. Most of the ACAT inhibitors currently available cause non-specific cytotoxicities and inhibit other enzymes with similar active sites, potentially causing other adverse side effects. The outcome of this application will help investigators to design ACAT1-specific inhibitors with minimal side effects, and help to determine the utility of ACAT1 as a target for treating atherosclerosis.