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.

Public Health Relevance

The long-term objective of this research is to understand the role of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in cellular cholesterol homeostasis. ACAT1 is a potential drug target for treating Alzheimer disease and other diseases in humans. The outcome of this application will help investigators to design ACAT1-specific inhibitors with minimal adverse side effects.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL060306-14A1
Application #
8295057
Study Section
Integrative Nutrition and Metabolic Processes Study Section (INMP)
Program Officer
Srinivas, Pothur R
Project Start
1998-04-01
Project End
2016-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
14
Fiscal Year
2012
Total Cost
$395,000
Indirect Cost
$145,000
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
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
Hu, Guang-Jing; Chen, Jia; Zhao, Xiao-Nan et al. (2013) Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene. Cell Res 23:1007-24
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
Rogers, Maximillian A; Liu, Jay; Kushnir, Mark M et al. (2012) Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase. J Biol Chem 287:17483-92
Chang, Catherine C Y; Miyazaki, Akira; Dong, Ruhong et al. (2010) Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy. Biochemistry 49:9957-63
Bryleva, Elena Y; Rogers, Maximillian A; Chang, Catherine C Y et al. (2010) ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD. Proc Natl Acad Sci U S A 107:3081-6
Sakashita, Naomi; Chang, Catherine C Y; Lei, Xiaofeng et al. (2010) Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity. J Lipid Res 51:1263-72
Lei, Lei; Xiong, Ying; Chen, Jia et al. (2009) TNF-alpha stimulates the ACAT1 expression in differentiating monocytes to promote the CE-laden cell formation. J Lipid Res 50:1057-67
Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C Y et al. (2009) Acyl-coenzyme A:cholesterol acyltransferases. Am J Physiol Endocrinol Metab 297:E1-9
Reid, Patrick C; Lin, Song; Vanier, Marie T et al. (2008) Partial blockage of sterol biosynthesis with a squalene synthase inhibitor in early postnatal Niemann-Pick type C npcnih null mice brains reduces neuronal cholesterol accumulation, abrogates astrogliosis, but may inhibit myelin maturation. J Neurosci Methods 168:15-25

Showing the most recent 10 out of 31 publications