Short - chain ubiquitination is associated with the degradation rate of a cell - surface - resident bile salt export pump ( BSEP / ABCB

This article has not been copyedited and formatted. The final version may differ from this version. Abstract 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 previously demonstrated that 4-phenylbutyrate (4PBA) treatment, an FDA-approved drug for urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. Conversely, 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. Accordingly, to help the development of the medical treatment of cholestasis, the present study 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 weight of the mature form of BSEP/Bsep shifted from 170 kDa to 190 kDa following ubiquitin modification (molecular weight: 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. This article has not been copyedited and formatted. The final version may differ from this version.