Department of Pharmaceutical Sciences, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA.
Department of Pharmaceutical Sciences, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA.
Biochim Biophys Acta Biomembr. 2021 Feb 1;1863(2):183510. doi: 10.1016/j.bbamem.2020.183510. Epub 2020 Nov 13.
The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (J) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.
人类顶端钠依赖性胆汁酸转运蛋白（hASBT，SLC10A2）是胆汁酸肠肝循环系统中肠道胆汁酸吸收的限速步骤。因此，hASBT 的调节和稳定性对于维持胆汁酸和胆固醇的体内平衡至关重要，并且可能成为与胆固醇相关疾病的潜在靶点。我们假设，调节 hASBT 功能和调节的翻译后机制将为肠道胆汁酸转运和体内平衡提供新的见解。在这项研究中，我们通过 COS-1 细胞中的酰基生物素交换确认了 hASBT 的 S-酰化状态及其对 hASBT 表达、功能、动力学和蛋白质稳定性的影响。使用酰化抑制剂 2-溴棕榈酸，我们表明 S-酰化是一种重要的修饰，调节 hASBT 的功能、表面表达和最大转运体通量（J）。通过蛋白酶体抑制剂，我们发现 S-酰化的 hASBT 通过蛋白酶体被清除，而 hASBT 的棕榈酰化状态减少会导致其比未修饰的转运体更快地发生蛋白水解降解。对跨膜结构域内或附近的半胱氨酸突变体进行筛选，其中一些暴露于细胞质中，证实 Cys314 是主要的 S-酰化残基。最后，我们表明，缺乏细胞质面向酪氨酸残基的 hASBT 突变体中的 S-酰化减少，提示翻译后修饰机制中的酰化和磷酸化之间存在串扰。
