Hayashi Hisamitsu, Sugiyama Yuichi
Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Mol Pharmacol. 2009 Jan;75(1):143-50. doi: 10.1124/mol.108.049288. Epub 2008 Oct 1.
The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We demonstrated previously that 4-phenylbutyrate (4PBA) treatment, a U.S. Food and Drug Administration-approved drug for the treatment of urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. On the other hand, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surface-resident BSEP. Therefore, to help the development of the medical treatment of cholestasis, we investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on short-chain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular mass of the mature form of BSEP/Bsep shifted from 170 to 190 kDa after ubiquitin modification (molecular mass, 8 kDa). Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations E297G and D482G. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surface-resident BSEP by 4PBA treatment and PFIC2-type mutations.
胆小管膜上胆盐输出泵（BSEP/ABCB11）表达降低与胆汁淤积诱导的肝毒性相关，这是由于胆汁酸在肝细胞中蓄积所致。我们之前证明，4-苯丁酸盐（4PBA）治疗（一种美国食品药品监督管理局批准用于治疗尿素循环障碍的药物）通过延长细胞表面驻留BSEP的降解速率来诱导BSEP的细胞表面表达。另一方面，在2型进行性家族性肝内胆汁淤积症（PFIC2）中发现的BSEP突变E297G和D482G，通过缩短细胞表面驻留BSEP的降解速率而使其表达降低。因此，为了推动胆汁淤积症治疗方法的发展，我们研究了4PBA和PFIC2型突变影响细胞表面BSEP降解的潜在机制，重点关注短链泛素化。在表达BSEP的Madin-Darby犬肾II（MDCK II）细胞和大鼠胆小管膜囊泡中，泛素修饰后（分子量8 kDa），成熟形式的BSEP/Bsep分子量从170 kDa变为190 kDa。4PBA处理在体外和体内均降低了BSEP/Bsep的泛素化敏感性，相反，BSEP突变E297G和D482G增强了其泛素化敏感性。此外，使用MDCK II细胞进行的生物素标记研究表明，将泛素与BSEP融合的细胞表面驻留嵌合蛋白的降解速度比BSEP本身更快。总之，BSEP/Bsep被两到三个泛素修饰，其泛素化受4PBA处理和PFIC2型突变调节。短链泛素化的调节可通过4PBA处理和PFIC2型突变来调控细胞表面驻留BSEP降解速率的变化。
