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Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.

ABSTRACT: ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys(75) and Cys(309)) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys(1477) was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys(1463) and Cys(1465), impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys(75), Cys(309), Cys(1463), Cys(1465), and Cys(1477)) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys(75) and Cys(309) in ECD1 and either Cys(1463) or Cys(1465) and Cys(1477) in ECD2) are required for ABCA1 to be fully functional.

SUBMITTER: Hozoji M

PROVIDER: S-EPMC2670134 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.

Hozoji Masako M Kimura Yasuhisa Y Kioka Noriyuki N Ueda Kazumitsu K

The Journal of biological chemistry 20090303 17

ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formatio ...[more]

PMID: 19258317

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Project description:Introduction: Atherosclerosis is the main cause of many cardiovascular diseases and contributes to morbidity and mortality worldwide. The formation of macrophage-derived foam cells plays a critical role in the early stage of atherosclerosis pathogenesis. Diterpenoids found in the flowers of Callicarpa rubella Lindl., a traditional Chinese medicine, have been reported to have anti-inflammatory activity. However, little is known about the effects of these diterpenoids on macrophage foam cell formation. Methods: A macrophage-derived foam cell formation model was established by treating RAW264.7 cells with oxidized low-density lipoprotein (ox-LDL) for 24 h. Oil red O staining were used to detect the intracellular lipids. The cholesterol efflux capacity was assayed by labeling cells with 22-NBD-cholesterol. Western blots and real-time PCRs were performed to quantify protein and mRNA expressions. Results: Two diterpenoid molecules, 14α-hydroxyisopimaric acid (C069002) and isopimaric acid (C069004), extracted from the flowers of Callicarpa rubella Lindl., significantly attenuated ox-LDL-induced foam cell formation in RAW264.7 macrophages. Further investigation showed that these two diterpenoids could promote cholesterol efflux from RAW264.7 macrophages to apolipoprotein A-I or high-density lipoproteins, which was associated with upregulated expression of ATP-binding cassette A1/G1 (ABCA1/G1), liver X receptor-α (LXRα), and peroxisome proliferator-activated receptor-γ (PPARγ). Unexpectedly, the diterpenoids C069002 and C069004 failed to enhance the mRNA transcription of the ABCG1 gene in macrophage-derived foam cells induced by ox-LDL. To evaluate the effects of diterpenoids on macrophage foam cell formation and determine the underlying mechanism, two drugs (lovastatin and rosiglitazone) were used as positive controls. Although both drugs could reduce macrophage foam cell formation and promote cholesterol efflux, they each had distinctive abilities to modulate the expression of cholesterol efflux-related genes. In contrast to lovastatin, rosiglitazone showed a similar influence on the expression of cholesterol efflux-related genes (including ABCA1, LXRα, and PPARγ) as the diterpenoids regardless of the presence or absence of ox-LDL, implying a similar mechanism by which they may exert atheroprotective effects. Conclusion: Our research indicates that diterpenoids effectively inhibit ox-LDL-induced macrophage foam cell formation by promoting cholesterol efflux from macrophages via the PPARγ-LXRα-ABCA1 pathway. Further investigation of diterpenoids as potential drugs for the treatment of atherosclerosis is warranted.

Dataset Information

Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.

Publications

Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.

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