The objective of this research project is the functional characterization of the ABCA1 protein. Our main goals are to analyze the mechanism of action and regulation of ABCA1 and to identify its potential interaction with intracellular proteins. Mutations in the ABCA1 gene result in Tangier disease a genetic disorder characterized by an abnormal lipoprotein profile and the accumulation of cholesterol esters in various tissues. The pathophysiology of the association between cholesterol and atherosclerosis is thought to involve the cellular uptake and deposition of cholesterol. By removing cholesterol from the cells, the reverse cholesterol pathway provides protection for the artery wall against unwanted lipid deposition. The recent discovery that ABCA1 controls the cellular apolipoprotein-mediated lipid removal pathway suggests that ABCA1 plays a key role in the modulation of the reverse cholesterol transport. However, the exact nature of the substrate(s) and the physiological circumstances of this transport are still unknown. In order to characterize the biochemical properties of ABCA1, we use two basic expression systems for the high level expression of the human ABCA1 protein: the baculovirus-Sf9 insect cell system and a retrovirus based expression system for mammalian cells. Probing the wild-type and mutant forms of ABCA1 helps to identify substrates, inhibitors and regulatory agents of this protein. We aim to study the function and subcellular localization of ABCA1 protein in polarized mammalian cell lines stable expressing ABCA1. We also plan to investigate the interaction of the COOH-terminus of ABCA1 protein with PDZ proteins and the potential functional consequences of this binding.

Overexpression of human ABCA1 in Sf9 insect cells, functional analysis of human ABCA1 by direct transport measurements in isolated membrane vesicles using radiolabeled and fluorescent analogs of cholesterol and phospholipids. Determination of the nature of the molecule(s) transported by ABCA1. These experiments will also be done in the presence and absence of ApoA-1 to determine whether an acceptor is needed for the transport process.

Stable expression of ABCA1 protein in polarized mammalian cells using retrovirus transduction system, functional analysis and subcellular localization of ABCA1. Transport measurements in intact cells overexpressing ABCA1 will be applied testing fluorescent analogs of cholesterol and phospholipid substrates. Both flow-cytometry and single cell imaging methods will be used in these studies.

Biochemical investigation of binding between the COOH-terminus of ABCA1 protein and PDZ proteins. Studies on the potential role of this interaction in the function or localization of ABCA1 membrane protein.

Functional investigation of mutant forms of ABCA1 either with substitution known to inhibit other ABC transporters or with mutations known to cause serious dysfunction’s in lipid metabolism. Characterization of these mutants permit the elucidation of the substrate recognition and the mechanism of action of ABCA1 protein.