S-acylation is Crucial for ASBT-Mediated Bile Acid Uptake: Monitoring the Transport Activity in Real Time
thesisposted on 01.05.2021, 00:00 by Alexander L Ticho
Bile acids are a structurally diverse class of molecules that serve a wide variety of critical physiological functions. These compounds are synthesized in the liver and secreted into the small intestine, where they facilitate lipid absorption and serve as important signaling molecules. Approximately 95% of bile acids are reabsorbed in the intestine and returned to the liver, establishing their enterohepatic circulation. The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for maintaining bile acid enterohepatic circulation, as evidenced by the fact that ASBT knockout leads to a decrease in the bile acid pool size. ASBT defects or dysregulation may contribute to several disorders, including bile acid malabsorption and hypercholesterolemia. ASBT is rapidly regulated by a variety of post-translational mechanisms, including phosphorylation and modulation of its interaction with plasma membrane microdomains, which may represent adaptive responses to a rapidly changing intestinal milieu. Investigating the rapid regulation of ASBT is challenging, as traditional methods do not capture transporter activity in real time. Several recent studies have developed real time methods in order to measure various aspects of bile acid homeostasis, but none have been able to directly measure the activity of bile acid transporters such as ASBT. We hypothesized that a bioluminescence-based approach for assessing bile acid transporters would allow for real-time monitoring of transporter activity. To address this hypothesis, we designed a reporter system relying on a novel probe: cholic acid attached to luciferin via a disulfide-containing, self-immolating linker (CA-SS-Luc). We first validated its use for in vitro monitoring of bile acid transporter activity using HEK 293 cells transfected with ASBT or the hepatic bile acid transporter NTCP, along with firefly luciferase. We demonstrated that real-time bioluminescence production in living cells was proportional to the cellular expression of bile acid transporter. We also investigated CA-SS-Luc in mouse intestinal epithelial cells isolated from transgenic mice expressing firefly luciferase. Ileal enterocytes displayed significantly higher luminescence compared to jejunal enterocytes, indicating a transport process mediated by ileal ASBT. These data provide evidence for the suitability of this method to assess the acute regulation of ASBT activity in real time. In this regard, rapid regulation of membrane proteins relies on post-translational modification of specific amino acid residues. Recent evidence has demonstrated that several cysteine residues present in the ASBT protein are critical for proper transport function. Cysteine residues may be subject to S-acylation, a post-translational lipid modification involving covalent binding to fatty acids, which has been shown to regulate membrane transport proteins. Therefore, we hypothesized that ASBT is subject to S-acylation, and that this modification is important for transporter function and bile acid uptake. With regard to ASBT regulation, we demonstrated that ASBT is S-acylated in native intestinal tissues from mice, as well as human organ donors. In HEK 293 cells transfected with ASBT, inhibition of S-acylation rapidly reduced ASBT activity, indicating that S-acylation is important for ASBT function. We further showed that S-acylation of ASBT by unsaturated fatty acids reduced transporter function compared to saturated fatty acids, suggesting that the fatty acid attached to ASBT is functionally important. In conclusion, these studies have established an innovative method for assessing bile acid transporters in living cells. We have also identified and characterized a novel post-translational lipid modification of ASBT that may provide important insights into the regulation of intestinal bile acid absorption and the maintenance of bile acid homeostasis.