Current Research Projects

1. Structure-function analyses of ACAT.

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 lipoprotein synthesis and assembly, in dietary cholesterol absorption, and in the early stages of atherosclerosis. It is a pharmaceutical target for therapeutic intervention of hyperlipidemia and atherosclerosis. The first gene encoding the enzyme, designated as ACAT1, was identified through an expression cloning approach in this laboratory. Using recombinant DNA technology, we have purified this protein to homogeneity. Future investigations are directed to use the biochemical and biophysical approaches to investigate the molecular basis of the cholesterol-dependent ACAT allostery, to identify the enzyme active and regulatory sites, and to determine the mechanisms of various ACAT inhibitors. In addition, a similar but different gene, designated as the ACAT2 gene, was recently discovered by homologous cloning. The physiological roles of ACAT1 and ACAT2 in different tissues are being investigated in this laboratory. Recent evidence suggests that ACAT may also be a potential drug target for treating Alzheimer's disease. Alzheimer's disease is a major neurodegenerative disease that affects an estimated 4.5 million Americans and is becoming

increasingly common in the U.S. population. We are currently taking a mouse genetic approach to determine the pathophysiological role of ACAT in a mouse model for Alzheimer's disease.

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.