Calpain protease catalyzes PEST-dependent degradation of ABCA1. (a) Levels of ABCA1 and ABCA1delPEST in 293 cells, incubated with or without calpeptin (30 μg/ml) for 3 hours, as determined by Western blot analysis. (b) Cell surface ABCA1 or ABCA1delPEST in 293 cells with or without incubation with calpeptin (20 μg/ml) for 3 hours, as determined by biotinylation, immunoprecipitation with anti-FLAG antibody, and Western analysis with avidin–horseradish peroxidase. The data are representative of two experiments, each performed in triplicate, with similar results. (c and d) The susceptibility to exogenously added μ-calpain of ABCA1 or ABCA1delPEST metabolically labeled by [³⁵S]methionine in digitonin-permeabilized 293 cells with (d) or without (c) pretreatment with apoA-I (10 μg/ml) for 3 hours.

ApoA-I–mediated increase in ABCA1 requires PEST sequence. (a) Dose-response effect of apoA-I (3 hours’ incubation) in HEK293 cells transfected with wild-type ABCA1. (b) Wild-type ABCA1 and ABCA1delPEST protein levels after 3 hours of incubation with apoA-I (10 μg/ml) in 293 cells transiently transfected with the FLAG-tagged constructs, as determined by Western blot with FLAG antibodies. The data are representative of three (a) or two (b) experiments with similar results.

ApoA-I and apoE increase ABCA1 protein in mouse primary hepatocytes and macrophages. (a) Time course of the effect of apoA-I (10 μg/ml) on ABCA1 protein levels in mouse peritoneal macrophages pretreated overnight with 50 μg/ml acetyl-LDL and LXR/RXR ligands 22(R)-hydroxycholesterol and 9-cis retinoic acid (RA) (both 10 μM). (b) Cell surface biotinylated ABCA1 levels in macrophages after 3 hours of incubation with 10 μg/ml apoA-I. (c) ABCA1 levels in primary mouse hepatocytes incubated for 3 hours with 10 μg/ml apoA-I. (d) Effects of apoA-I (10 μg/ml) on ABCA1 levels in macrophages treated with or without acetyl-LDL (AcLDL) and LXR/RXR ligands. (e) Dose response of apoE₃ effect (3 hours’ incubation) on ABCA1 protein levels in mouse peritoneal macrophages pretreated with acetyl-LDL, 22(R)-hydroxycholesterol, and 9-cis retinoic acid. (f) ABCA1 levels in mouse peritoneal macrophages after 3 hours of incubation with or without apoE₃ (10 μg/ml). (g) ABCA1, W590S-ABCA1, and ABCA1delPEST protein levels in HEK293 cells after 3 hours of incubation with or without apoE₃. The data are representative of two (a, b, e, and g) or three (c, d, and f) experiments with similar results.

A PEST sequence regulates the cell surface expression and function of ABCA1. (a) Schematic model of ABCA1 showing the location and amino acid sequence of the PEST sequence in mouse (m), human (h), and chicken (c). The PEST score is +16.42, as determined by PESTfind. (b) Levels of total (bottom) and cell surface (top, biotinylated) ABCA1 and ABCA1delPEST in transiently transfected HEK293 cells. (c, f, and g) DNA dose-dependent ABCA1-mediated and ABCA1delPEST-mediated cellular cholesterol efflux (c), phospholipid efflux (f), and apoA-I cell association (g). (d) Total cellular ABCA1 protein mass as determined by quantitative Western blotting in the cholesterol efflux assay. (e) Cholesterol efflux efficiency determined by percentage efflux normalized for total cellular ABCA1 protein mass. (h) Levels of total (bottom) and ubiquitinated (top) ABCA1 and ABCA1delPEST in HEK293 cells incubated with or without lactacystin (20 μM) for 3 hours. The data are representative of two experiments (c and e–h, triplicate; d, duplicate) or three experiments (b, duplicate and triplicate) with similar results.

Calpeptin increases ABCA1 in primary mouse macrophages and hepatocytes. Levels of ABCA1 in mouse peritoneal macrophages (a) or primary mouse hepatocytes (b) incubated with or without calpeptin at indicated concentration for 3 hours. The data are representative of three independent experiments with similar results.

Effects of cholesterol and phospholipid efflux on the induction of ABCA1 by apoA-I. (a, top) ABCA1 protein level in mouse peritoneal macrophages after 3 hours of incubation with 10 μg/ml apoA-I or 50 μg/ml HDL₂, or after pretreatment with 1 mM methyl-β-cyclodextrin (MβCD) for 5 minutes to deplete cholesterol and then with control media for 3 hours. (a, bottom) Phospholipid (Pl) and cholesterol (Ch) efflux in macrophages treatments similar to those in the top panel. (b) ABCA1-FLAG and Walker-mutant (K939M) ABCA1-FLAG (ABCA1WM) protein levels after 3 hours of incubation with apoA-I (10 μg/ml) in transiently transfected 293 cells. (c) Wild-type ABCA1-FLAG and ABCA1-W590S-FLAG protein levels after 3 hours of incubation with apoA-I (10 μg/ml) or calpeptin (20 μg/ml) in 293 cells. The data are representative of two (a and b) or three (c) experiments with similar results.

ApoA-I injection increases ABCA1 protein in hepatocytes and macrophages in mice. (a) Hepatic ABCA1 levels in mice 4 hours after intravenous injection of apoA-I (20 mg/kg body weight) or albumin as control (Ctr). The bar graph represents quantification of ABCA1 protein levels normalized against β-actin (n = 5 for each group, P < 0.01). (b) Macrophage ABCA1 levels in mice 4 hours after apoA-I injection (n = 5 for apoA-I group and n = 3 for control group, P < 0.006).