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.
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