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

Abstract

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.

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications

“…The novel S- palmitoylation on Glut4 and IRAP was confirmed, and altered levels of their S- palmitoylation may play a role in obesity . Similarly, S- palmitoylation was validated and found to modulate the function of the apical sodium-dependent bile acid transporter (ASBT) crucial for the circulation of bile acids in human cells . Moreover, the S- palmitoylation on the C-terminal region of mucolipin 3 whose activation induces autophagy in a S- palmitoylation-dependent manner was verified .…”

“…256 Similarly, S-palmitoylation was validated and found to modulate the function of the apical sodium-dependent bile acid transporter (ASBT) crucial for the circulation of bile acids in human cells. 257 Moreover, the S-palmitoylation on the C-terminal region of mucolipin 3 whose activation induces autophagy in a Spalmitoylation-dependent manner was verified. 258 This approach also aided in the discovery of the regulatory function of dickkopf1 (DKK1) in the dynamic S-palmitoylation of its receptors CKAP4 and LRP6, which may play pivotal roles in cancer proliferation.…”

Regulation of Drug Transport Proteins—From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium

“…Mutagenesis of the cysteine residues in OATP2B1 also led to misprocessing and intracellular accumulation in CHO‐OATP2B1 cells 62 . Similarly, S ‐acylated ASBT, the predominant form in ileal brush border membrane vesicles derived from human donor ileum, is important for activity, plasma membrane localization, and protection from degradation 63 . Localization of transporters like P‐gp and BCRP in plasma membrane “lipid rafts” impacts efflux activity 64 ; for example, depletion of cellular cholesterol content by methyl‐beta‐cyclodextrin caused a 40% decrease in BCRP activity in MDCKII‐BCRP cells 64 .…”

“…62 Similarly, Sacylated ASBT, the predominant form in ileal brush border membrane vesicles derived from human donor ileum, is important for activity, plasma membrane localization, and protection from degradation. 63 Localization of transporters like P-gp and BCRP in plasma membrane "lipid rafts" impacts efflux activity 64 ; for example, depletion of cellular cholesterol content by methylbeta-cyclodextrin caused a 40% decrease in BCRP activity in MDCKII-BCRP cells. 64 The activation of PKC accelerated OATP2B1 internalization via the clathrin-dependent pathway and subsequent lysosomal degradation in MDCKII-OATP2B1 and Caco-2 cell monolayers.…”

Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

“…[5] As a result, major efforts have gone into applying bioorthogonal chemistry to the study of lipid biochemistry. By introducing small terminal alkynes and azides in fatty acid tails [6][7][8][9][10][11] , phospholipids [12,13] , sphingolipids [14] , and cholesterol [15][16][17] , it has been possible to study lipids with only minimal modifications compared to the endogenous molecules. This strategy reduces the chances of the modifications affecting the native function of the lipid, and has successfully been used to study lipid localisation [10,12,13,17] , metabolism [11,16] , and trafficking [18][19][20] , as well as post-translational lipidation of proteins (reviewed by Distefano and co-workers [21] ).…”