STRUCTURE/FUNCTION ANALYSIS OF ACAT

这个实验室的长期研究兴趣是了解 胆固醇代谢的细胞和分子调控 水平。酰基辅酶A：胆固醇酰基转移酶(ACAT)被认为 在饮食胆固醇吸收中发挥重要作用，在肝脏 脂蛋白的组装和分泌，以及在产生泡沫细胞中的作用 动脉粥样硬化斑块。申请人的实验室最近 提出ACAT可能是一种变构酶，作为一种 内质网中的胆固醇传感器；ACAT与内质网胆固醇的结合可能 在ACAT中引起配体诱导的结构/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。配体诱导的构象变化可能通过以下途径发生 ACAT全酶中多个亚基之间的相互作用。这个 申请人的实验室最近成功地净化了 重组人ACAT蛋白在CHO细胞中表达达到均一。 我们还合成了一种新的耐光性胆固醇类似物。使用 这些可用的分子试剂，在目前的提案中，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ACAT的有效性 变构调节模型，并阐明变构调节的分子基础 胆固醇和ACAT之间的传感机制。 我们的具体目标是：1.确定ACAT在ER中的拓扑 薄膜。2.研究均相ACAT的性质。 重组的PC囊泡。3.鉴定胆固醇结合 光亲和标记ACAT内的位点(S)和位点特异性 诱变。4.制备由以下组成的ACAT杂低聚物 活性的ACAT单体和非活性的ACAT单体，并检测 它们在体外的特性。5.研究低聚物的结构 ACAT在体外和在完整的CHO细胞中。

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.