S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells

• Published in: The Journal of biological chemistry 2020-04, Vol.295 (14), p.4488-4497
• Main Authors: Ticho, Alexander L., Malhotra, Pooja, Manzella, Christopher R., Dudeja, Pradeep K., Saksena, Seema, Gill, Ravinder K., Alrefai, Waddah A.
• Format: Article
• Language: English
• Subjects: Acylation - drug effects; Acyltransferases - metabolism; Alkynes - chemistry; apical sodium-dependent bile acid transporter (ASBT); bile acid; bile acid transport; Bile Acids and Salts - metabolism; Cell Biology; Cell Membrane - metabolism; Cysteine - chemistry; Cysteine - metabolism; HEK293 Cells; Humans; ileum; Ileum - metabolism; intestinal epithelial cells; intestinal epithelium; intestine; lipid modification; membrane transport; Oleic Acid - chemistry; Oleic Acid - pharmacology; Organic Anion Transporters, Sodium-Dependent - genetics; Organic Anion Transporters, Sodium-Dependent - metabolism; Palmitates - chemistry; Palmitates - pharmacology; posttranslational modification (PTM); protein acylation; S-acylation; SLC10A2; Symporters - genetics; Symporters - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.011032

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (˜80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne–palmitic acid (100 μm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 μm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 μm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne–oleic acid (100 μm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT’s function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.