Helical Apolipoproteins Stabilize ATP-binding Cassette Transporter A1 by Protecting It from Thiol Protease-mediated Degradation

Abstract

ATP-binding cassette transporter (ABC) A1 was increased by apolipoprotein A-I without an increase of its message in THP-1 cells. The pulse label study demonstrated that apoA-I retarded degradation of ABCA1. Similar changes were demonstrated by apoA-II, but the effect of high density lipoprotein was almost negligible on the basis of equivalent protein concentration. Thiol protease inhibitors (leupeptin and N-acetyl-Leu-Leunorleucinal (ALLN)) increased ABCA1 and slowed its decay in the cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH 4 Cl showed such effects. The effects of apoA-I and ALLN were additive for the increase of ABCA1, and the apoA-I-mediated cellular lipid release was enhanced by ALLN. The data suggest that ABCA1 is rapidly degraded by a thiol protease(s) in the cells unless helical apolipoproteins in their lipid-free form stabilize ABCA1 by protecting it from proteasemediated degradation.Helical apolipoproteins such as apoA-I, apoA-II, apoA-IV, and apoE interact with cell surfaces and generate high density lipoprotein (HDL) 1 by removing cellular phospholipid and cholesterol (1-3). The reaction is recognized as one of the major pathways of cellular cholesterol release along with the diffusion-mediated nonspecific cholesterol efflux (4). Fibroblasts from patients with a genetic defect of plasma HDL lack the interaction with apolipoprotein, indicating that this reaction is a main source of plasma HDL (5, 6). This view is supported by the finding that probucol, which markedly reduces plasma HDL, blocks the cell-apolipoprotein interaction (7-9). Mutations were identified in ATP-binding cassette transporter (ABC) A1 with many HDL-deficient families (10 -12), so that this protein is considered as a key for generation of HDL by the apolipoprotein-cell interaction and release of cellular cholesterol by this pathway.Regulation of ABCA1 expression is primarily by cellular cholesterol level through oxysterol as a ligand for the nuclear receptor liver X receptor (LXR) that acts in a heterodimer with retinoid X receptor (RXR) (13-15). The ligands for RXR (such as retinoids) in this receptor system also up-regulate the ABCA1 gene expression (14,15). In certain types of cell such as RAW264, cyclic AMP and its analogues markedly increase the ABCA1 message and protein by an unknown mechanism (16, 17). In THP-1 cells, differentiation by phorbol ester induces an increase in ABCA1 message level (18). ABCA1 was found in early and late endosomes so that the lysosomal pathway was suggested for its degradation (19). More recently, proteolytic degradation was indicated as a factor involved in regulation of the cellular level of ABCA1 (20). EXPERIMENTAL PROCEDURESCell Culture-THP-1 cells were maintained in RPMI 1640 (Iwaki) containing 10% fetal bovine serum (PAA laboratories) in a humidified atmosphere of 5% CO 2 and 95% air. Differentiation of THP-1 monocytes into macrophages was induced by culturing the cells at a density of 3.0 ϫ 106 cells/well ...

Effects of Apolipoprotein A-I on ATP-binding Cassette Transporter A1-mediated Efflux of Macrophage Phospholipid and Cholesterol

“…Stimulation of the cells with cAMP increased the release of this material by ϳ60% over 6 h relative to unstimulated cells incubated in the absence of apoA-I (data not shown). Similarly, the presence of apoA-I (5 g/ml for 6 h) enhanced the release of this void volume material by ϳ3-fold (data not shown), perhaps due to the stabilizing effect of apoA-I on ABCA1 (52). Fig.…”

Paraoxonase 1‐treated oxLDL promotes cholesterol efflux from macrophages by stimulating the PPARγ–LXRα–ABCA1 pathway

“…A mutation of ABCA1 has been shown to abolish cholesterol efflux and increase the risk of developing atherosclerosis [18]. ABCA1 is regulated both at the transcriptional level via liver X receptor (LXR)/retinoid X receptor-mediated induction of the ABCA1 gene [19] and at the post-translational level via changes in the turnover of the ABCA1 protein [20]. ABCA1 expression is induced by PPARc through a transcriptional cascade mediated by the nuclear receptor LXRa [21].…”

Probucol Inactivates ABCA1 in the Plasma Membrane with Respect to Its Mediation of Apolipoprotein Binding and High Density Lipoprotein Assembly and to Its Proteolytic Degradation

“…Western Blotting-The membrane fraction and cellular subfractions were resuspended in 50 mM Tris-HCl (pH 7.5) containing 5 mM EDTA, 10 mM EGTA, 1 mM PMSF, 1 mM benzamidine, 1% Triton X-100, and 1% protease inhibitor cocktails (Sigma) and were sonicated for 5 s. After determination of the protein content by a BCA method (Pierce), the fractions were dissolved in 9 M urea, 2% Triton X-100, 1% dithiothreitol and were developed in 6 or 15% (w/v) polyacrylamide gel electrophoresis in the presence of 10% SDS, respectively, and the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) by semidry blotter in blotting buffer (25 mM Tris-HCl, 0.2 M glycine, and 10% methanol (v/v)) for 3.5 h. The membrane was blocked with 5% skim milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20 and was probed with the rabbit antiserum against the C-terminal peptide of human ABCA1 (26,39), rabbit anticaveolin-1 (N-20) (Santa Cruz Biotechnology), anti-GLUT-1, mouse anti-integrin ␤ 1 (CHEMICON International, Inc.), anti-GM130, and anti-Bip/GRP78 (BD Transduction Laboratories, BD Biosciences), respectively. The immunoreactive proteins were visualized by ECL or the ECL Plus system (Amersham Biosciences).…”