Functional Analysis of ACAT

ACAT的功能分析

基本信息

批准号：
8644841
负责人：
Ta Yuan CHANG
金额：
$ 44.44万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8644841
关键词：
Abbreviations Active Sites Acyl Coenzyme A Acyltransferase Adverse effects Affect Alzheimer&apos s Disease Amino Acids Apolipoprotein E Atherosclerosis Binding Biochemical Biological Assay Bone Marrow Catalysis Cells Chinese Hamster Cholesterol Cholesterol Esters Cholesterol Homeostasis Data Disease Drug Targeting Endoplasmic Reticulum Enzymes Family Foam Cells Genes Goals Health Hematopoietic stem cells Human In Vitro Integral Membrane Protein Isoenzymes Knock-out Leukocytosis Lipid Bilayers Low Density Lipoprotein Receptor Membrane Modeling Monitor Morbidity - disease rate Mus Mutation Neurodegenerative Disorders Outcome Ovary Pharmaceutical Preparations Physiological Play Procedures Proteins Reagent Recombinants Research Research Personnel Research Proposals Role Site-Directed Mutagenesis Staging Stem cells Sterol O-Acyltransferase Structure Tissues Transgenic Mice Transmembrane Domain apolipoprotein B-48 base cell type cytotoxicity design enzyme activity in vivo inhibitor/antagonist macrophage monocyte mortality mouse model novel overexpression recombinase research study sterol O-acyltransferase 1 sterol O-acyltransferase 2 tool

项目摘要

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.

描述（由申请人提供）：本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中的作用，动脉粥样硬化是全世界发病和死亡的主要原因。目前的提案有两个主要目标：继续在体外进行ACAT1的结构功能分析，并在Apoe-/-小鼠模型和Ldlr-/- apoB48缺陷小鼠模型中检查体内巨噬细胞ACAT1的作用用于动脉粥样硬化。作为细胞胆固醇代谢中的关键酶，ACAT 利用胆固醇和长链脂肪酰辅酶 A 产生胆固醇酯，并被其自身底物胆固醇变构激活，以防止胆固醇在内质网中过量积聚。 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发挥关键作用。 ACAT1 也在许多其他细胞类型中表达，包括骨髓中的造血干细胞。 ACAT1 在各种组织中的生理作用仍有待仔细研究。因此，该项目的目的是 1) 鉴定参与 ACAT1 底物结合、催化和/或变构控制的关键氨基酸残基； 2) 描述巨噬细胞 Acat1 失活在动脉粥样硬化起始和进展阶段的影响。我们将采用新的重组 hACAT1 酶表达和纯化程序以及七种不同的生化测定来实现目标 1，并将采用新开发的巨噬细胞特异性 Acat1 KO 小鼠 (Acat1-M/-M) 模型作为新型试剂追求目标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 抑制剂会引起非特异性细胞毒性，并抑制具有相似活性位点的其他酶，可能导致其他不良副作用。该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效用。