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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL060306-01
Application #
2635386
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Wang, Yong-Jian; Bian, Yan; Luo, Jie et al. (2017) Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation. Nat Cell Biol 19:808-819
Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan (2017) Methods for Monitoring ABCA1-Dependent Sterol Release. Methods Mol Biol 1583:257-273
Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan (2016) ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis. J Lipid Res 57:77-88
Huang, Li-Hao; Melton, Elaina M; Li, Haibo et al. (2016) Myeloid Acyl-CoA:Cholesterol Acyltransferase 1 Deficiency Reduces Lesion Macrophage Content and Suppresses Atherosclerosis Progression. J Biol Chem 291:6232-44
Yang, Wei; Bai, Yibing; Xiong, Ying et al. (2016) Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism. Nature 531:651-5
Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang et al. (2015) Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators. J Steroid Biochem Mol Biol 151:102-7
Yamauchi, Yoshio; Iwamoto, Noriyuki; Rogers, Maximillian A et al. (2015) Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum. J Biol Chem 290:23464-77
Poirier, Steve; Samami, Samaneh; Mamarbachi, Maya et al. (2014) The epigenetic drug 5-azacytidine interferes with cholesterol and lipid metabolism. J Biol Chem 289:18736-51
Huang, Li-Hao; Nishi, Koji; Li, Song et al. (2014) Acyl-coenzyme A:cholesterol acyltransferase 1 - significance of single-nucleotide polymorphism at residue 526 and the role of Pro347 near the fifth transmembrane domain. FEBS J 281:1773-83
Huang, Li-Hao; Gui, Jingang; Artinger, Erika et al. (2013) Acat1 gene ablation in mice increases hematopoietic progenitor cell proliferation in bone marrow and causes leukocytosis. Arterioscler Thromb Vasc Biol 33:2081-7

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