STRUCTURE/FUNCTION ANALYSIS OF ACAT

ACAT的结构/功能分析

• 批准号: 2635386
• 负责人: Ta Yuan CHANG
• 金额: $ 30.82万
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2635386
• 关键词: CHO cells; active sites; acyltransferase; affinity labeling; allosteric site; blood lipoprotein metabolism; cholesterol; conformation; endoplasmic reticulum; enzyme structure; fluorescence microscopy; immunofluorescence technique; ligands; protein binding; protein purification; protein structure function; recombinant proteins; site directed mutagenesis

The long-term research interest of this laboratory is to understand regulation of cholesterol metabolism at the cellular and molecular level. Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is believed to play important roles in dietary cholesterol absorption, in hepatic lipoprotein assembly and secretion, and in producing foam cells in atherosclerotic plaques. The applicant's laboratory has recently proposed that ACAT may be an allosteric enzyme and serves as a cholesterol sensor in the ER; binding of ER cholesterol by ACAT may cause a ligand induced structural/configuration change in the ACAT protein, which converts the enzyme from an inactive form to an active form. The ligand-induced configurational change may occur through interactions among multiple subunits in the ACAT holoenzyme. The applicant's laboratory very recently has succeeded in purifying the recombinant human ACAT protein expressed in CHO cells to homogeneity. We have also synthesized a new photolabile cholesterol analog. With these molecular reagents available, in the current proposal, we propose to use the recombinant DNA technology and biochemical methods to conduct structure-function analysis of ACAT, to test the validity of the allosteric regulation model, and to elucidate the molecular basis of the sensing mechanism between cholesterol and ACAT. Our specific aims are: 1. To determine the topology of ACAT in the ER membrane. 2. To study properties of the homogeneous ACAT in reconstituted PC vesicles. 3. To identify the cholesterol binding site(s) within ACAT by photoaffinity labelling and by site-specific mutagenesis. 4. To produce ACAT hetero-oligomeric complex composed of the active ACAT monomer and the inactive ACAT monomer, and to examine their properties in vitro. 5. To examine the oligomeric structure of ACAT in vitro and in intact CHO cells.

该实验室的长期研究兴趣是了解 细胞和分子水平上胆固醇代谢的调节 等级。 酰基辅酶 A：胆固醇酰基转移酶 (ACAT) 被认为 在膳食胆固醇吸收、肝脏中发挥重要作用 脂蛋白组装和分泌，以及产生泡沫细胞 动脉粥样硬化斑块。 申请人实验室近期 提出 ACAT 可能是一种变构酶并充当 ER 中的胆固醇传感器； ACAT 与 ER 胆固醇的结合可能 引起配体诱导的 ACAT 结构/构型变化 蛋白质，它将酶从非活性形式转化为活性形式 形式。 配体诱导的构型变化可能通过以下方式发生 ACAT 全酶中多个亚基之间的相互作用。 这 申请人的实验室最近成功纯化了 重组人 ACAT 蛋白在 CHO 细胞中表达均一。 我们还合成了一种新的光不稳定胆固醇类似物。 和 这些分子试剂可用，在当前的提案中，我们建议 利用重组DNA技术和生化方法进行 ACAT的结构-功能分析，以检验该方法的有效性 变构调节模型，并阐明其分子基础 胆固醇和ACAT之间的传感机制。 我们的具体目标是： 1. 确定 ER 中 ACAT 的拓扑结构 膜。 2. 研究同质ACAT的性质 重组PC囊泡。 3. 鉴定胆固醇结合 通过光亲和标记和位点特异性确定 ACAT 内的位点 诱变。 4. 生产 ACAT 异源寡聚复合物，其组成为 活性ACAT单体和非活性ACAT单体，并检查 它们的体外特性。 5. 检查寡聚结构 体外和完整 CHO 细胞中的 ACAT。