No abstract

ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein. Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h. To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ (PSD95-Discs large-ZO1) proteins, ␣1-syntrophin and Lin7, were found to interact with ABCA1. Immunoprecipitation revealed that ␣1-syntrophin interacted with ABCA1 strongly and that the interaction was via the Cterminal three amino acids SYV of ABCA1. Co-expression of ␣1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing ␣1-syntrophin. This effect is not common among PDZcontaining proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1. Co-expression of ␣1-syntrophin significantly increased the apoA-I-mediated release of cholesterol. ABCA1 was co-immunoprecipitated with ␣1-syntrophin from mouse brain. These results suggest that ␣1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release.Cholesterol is not catabolized in the peripheral cells and, therefore, is mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may also remove cholesterol that has pathologically accumulated in cells, such as at the initial stage of atherosclerosis. The assembly of high density lipoprotein (HDL) 1 particles by lipid-free apolipoproteins with cellular lipid has been recognized as one of the major mechanisms for the cellular cholesterol release (1, 2). ApoA-I-mediated cholesterol efflux is a major event in "reverse cholesterol transport," a process that generates HDL and transports excess cholesterol from the peripheral tissues, including the arterial wall, to the liver for biliary secretion. The importance of ABCA1 in this active cholesterol-releasing pathway for regulating cholesterol homeostasis became apparent with the finding that it is impaired in the cells from patients with Tangier disease, a genetic deficiency of circulating HDL (3, 4). Tangier disease is caused by mutations in ABCA1. ABCA1 mutations are also a cause of familial HDL deficiency and are associated with premature atherosclerosis (5, 6). Cholesterol is a prerequisite for cells, but, at the same time, the hyper-accumulation of cholesterol is harmful to cells. Therefore, the expression of ABCA1 is highly regulated at both the transcriptional and post-transcriptional level. The transcription of ABCA1 is regulated by the intracellular oxysterol concentration via the LXR/RXR nuclear receptor (7), and the synt...

Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXR␤/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXR␤/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXR␤, and the LXR␤/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXR␤ can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXR␤ is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.Maintenance of cellular cholesterol homeostasis is important for normal human physiology. Disruption of cellular cholesterol homeostasis leads to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATPbinding cassette protein A1), one of the key proteins in cholesterol homeostasis, mediates secretion of cellular free cholesterol and phospholipids to an extracellular acceptor in the plasma, apoA-I, to form high density lipoprotein (HDL) 3 (2, 3). HDL formation is the only known pathway that can eliminate excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4 -6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1-mediated cholesterol efflux is highly regulated at both the transcriptional and post-transcriptional levels. When cholesterol accumulates in cells, intracellular concentrations of oxysterols increase; subsequently, the liver X receptor (LXR), activated via binding of oxysterols, stimulates the transcription of ABCA1 (8 -10). ABCA1 protein eliminates excess cellular cholesterol and turns over rapidly with a half-life of 1-2 h (11-15). Several proteins, including apoA-I, ␣1-syntrophin, and ␤1-syntrophin, have been reported to interact with ABCA1 and reduce the rate of ABCA1 protein degradation (13-16). Syntrophins play critical roles in regulating the apoA-I-dependent cholesterol efflux (and thus in lipid homeostasis) by suppressing protein degradation in brain (14) and liver (15). Because cholesterol is an essential component of cells, however, excessive elimination of cholesterol could result in cell death. Con...

Cells have evolved multiple mechanisms for maintaining cholesterol homeostasis, and, among these, ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux is highly regulated at the transcriptional level through the activity of the nuclear receptor liver X receptor (LXR). Here, we show that in addition to its well defined role in transcription, LXR␤ directly binds to the C-terminal region ( Disruption of cellular cholesterol homeostasis can lead to a variety of pathological conditions, including cardiovascular disease (1). ATP-binding cassette protein A1 (ABCA1), 2 an important regulator of cholesterol homeostasis, mediates the release of cellular excess free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor circulating in plasma, to form high density lipoprotein (HDL) (2-5). HDL formation is the only pathway through which excess cholesterol can be eliminated from nonhepatic cells. Defects in ABCA1 cause Tangier disease (6 -8), a condition in which patients have a near absence of circulating HDL, prominent cholesterol-ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 9).The ABCA1-mediated release of cholesterol is highly regulated at the transcriptional level. When excess cholesterol accumulates in cells, intracellular concentrations of oxysterols increase and activate liver X receptor (LXR), which, in turn, stimulates ABCA1 gene transcription and increased expression of ABCA1 with associated elimination of excess cholesterol (10 -12). However, cholesterol is required for cell function and proliferation, and the intracellular cholesterol concentration must be maintained within a narrow range. Consequently, ABCA1-mediated cholesterol release is also regulated at the post-translational level. Several proteins, including syntrophins (13, 14), JAK2 (15), and LXR␤ (16), have been reported to interact with ABCA1 and modulate its degradation and function. However, the precise mechanism(s) by which ABCA1 activity is regulated post-translationally remains unclear.We previously reported (16) that a fraction of cytosolically localized LXR␤ may interact with ABCA1 on the plasma membrane and modulate the function of ABCA1. In WI-38 and THP-1 cells, endogenous LXR␤ interacts with ABCA1 under conditions in which LXR ligands do not accumulate, i.e. when cholesterol is not in excess. LXR␤ suppresses ABCA1-mediated cholesterol efflux. However, the mechanism by which LXR␤ suppresses ABCA1 functions was not clear. In this study, we identified two leucine residues in the C-terminal region of ABCA1 responsible for the interaction with LXR␤ and showed that LXR␤ interaction suppresses ATP binding to ABCA1 and thereby keeps ABCA1 standby on the plasma membrane for acute cholesterol accumulation. EXPERIMENTAL PROCEDURESMaterials-The LXR ligand TO901317, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and Alexa Fluor 546 succinimidyl esters were purchased from Cayman Co., Sigma, Stelaroids, and Molecular Probes, respectively. A cholesterol oxidas...