1. Structure-function analyses of ACAT1 in vitro and in vivo.
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. This enzyme plays important roles in cellular cholesterol homeostasis and in atherosclerosis. It is a pharmaceutical target for therapeutic intervention of hyperlipidemia and atherosclerosis. The first gene encoding the enzyme, designated as ACAT1, was identified in this laboratory. We have expressed ACAT1 as a recombinant protein in mammalian cells and have purified it to homogeneity. We are currently taking biochemical and biophysical approaches to identify the enzymeís active and regulatory sites, and to determine the mechanisms of action of various ACAT inhibitors. Recent evidence has shown that ACAT1 is also a drug target for treating neurodegenerative diseases including Alzheimer's disease, which is a major neurodegenerative disease in the developed countries. We are currently taking mouse genetic and cell biological approaches to determine the pathophysiological role of ACAT1 in multiple human diseases including Alzheimer's disease, atherosclerosis, and diet-induced obesity.
2. To delineate various intracellular cholesterol trafficking steps.
The process of intracellular cholesterol trafficking is involved in many physiological events, including lipoprotein synthesis and secretion, steroidogenesis, cholesterol accumulation in macrophages, and synthesis and maintenance of neuronal cell membranes. Abnormalities in these events often lead to diseases in animals and humans. A clear example is the Niemann-Pick type C1 disease. Patients who carry the homozygous form of NPC1 rarely live beyond their teenage years. In mammalian cells, low density lipoprotein (LDL) binds to its receptor, internalizes and enters the endosomes/lysosomes for hydrolysis of the lipid cargo cholesteryl esters. In fibroblast cells isolated from NPC1 patients, it has been shown that cholesterol derived from LDL cannot be delivered to various destinations; instead, it accumulates intracellularly. Using CHO cells mutated at the NPC1 locus and other loci as tools, we are studying the intracellular cholesterol trafficking in CHO cells and in other cell types, with the goal of delineating each discrete step at the molecular level.
Cholesterol Sensing, Trafficking, and Esterification