ABCMdb

PMID: 18829893

Hayashi H, Sugiyama Y
Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11).
Mol Pharmacol. 2009 Jan;75(1):143-50. Epub 2008 Oct 1., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 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. Login to comment 5 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 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. Login to comment 20 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 1 Copyright (c) 2009 The American Society for Pharmacology and Experimental Therapeutics 49288/3415636 Mol Pharmacol 75:143-150, 2009 Printed in U.S.A. degradation from the endoplasmic reticulum (ER) are responsible for the reduced cell-surface expression of BSEP in PFIC2 patients with E297G and D482G mutations (Hayashi and Sugiyama, 2007), both of which are the most frequently found in patients with PFIC2 (Strautnieks et al., 2008). Login to comment 42 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The BD Adeno-X Adenoviral Expression System (BD Biosciences) was used to create BSEP, E297G BSEP, D482G BSEP, HA-BSEP, HA-BSEP-Ub⌬GG , and HA-BSEP-Ub⌬GG/I44A recombinant adenoviruses as described previously (Hayashi et al., 2005a). Login to comment 60 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly After a 24-h culture, confluent cells were infected with recombinant adenovirus containing cDNAs for BSEP, E297G BSEP, D482G BSEP, HA-BSEP, and GFP at a multiplicity of infection of 200. Login to comment 112 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We reported previously that E297G and D482G, frequent mutations in PFIC2 patients, shorten the half-life of cell-surface-resident BSEP by approximately 1.5-and 4-fold, respectively, and, conversely, 4PBA treatment prolongs cell-surface-resident BSEP 2-fold (Hayashi and Sugiyama, 2007). Login to comment 113 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To explore a possible correlation between the half-life of cell-surface-resident BSEP and the short-chain ubiquitination susceptibility of BSEP, mutated BSEP was immunoprecipitated from MDCK II cells expressing E297G BSEP and D482G BSEP, and the immunoprecipitates were subjected to Western blot analysis for Ub and BSEP (Fig. 2A). Login to comment 114 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly BSEP, E297G BSEP, and Bsep were also immunoprecipitated from MDCK II cells expressing BSEP and E297G BSEP after 4PBA treatment and rCMVs prepared from 4PBA-treated SD rats, and the immunoprecipitates were subjected to Western blot analysis for Ub and BSEP (Fig. 3, A-C). Login to comment 115 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Quantitative densitometry analysis revealed that the ratio of the short-chain ubiquitinated BSEP, PFIC2-type mutated BSEPs (Figs. 2A and 3, A and B, arrow) to the mature form of BSEP, PFIC2-type mutated BSEPs (Figs. 2A and 3, A and B, filled arrowhead) was significantly greater, 6- and 30-fold by E297G and D482G mutations, respectively, than that in wild-type BSEP (Fig. 2B), and was reduced in a time-dependent manner after 4PBA treatment in vitro (Fig. 3, D and E). Login to comment 120 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly A, short-chain ubiquitination susceptibility of PFIC2-type mutated BSEPs, E297G BSEP and D482G BSEP. Login to comment 126 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Open, gray, and closed columns represent the ratio of band density indicating the short-chain ubiquitinated BSEP to that indicating the mature form of BSEP in MDCK II cells expressing BSEP, E297G BSEP, and D482G BSEP, respectively. Each bar represents the mean Ϯ S.E., n ϭ 3 to 4. Asterisks represent statistically significant differences between BSEP and mutated BSEP, ‫,ء‬ P Ͻ 0.05, and ‫,ءء‬ P Ͻ 0.01. Login to comment 136 ABCB11 p.Glu297Gly A and B, short-chain ubiquitination susceptibility of BSEP (A) and E297G BSEP (B) in 4PBA-treated MDCK II cells. Login to comment 141 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Bsep was immunoprecipitated from solubilized rCMVs prepared from 4PBA-treated SD rats with anti-rBsep antibody. Immunoprecipitates were separated by 6% SDS-PAGE and subjected to Western blot analysis. D and E, quantification of the short-chain ubiquitinated BSEP and E297G BSEP normalized with regard to the mature form of BSEP and E297G BSEP in A and B. Login to comment 148 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We have found previously that shortening the half-life of cell-surface-resident BSEP is partly responsible for the reduced cell surface expression of BSEP in patients with PFIC2 with E297G and D482G mutations (Hayashi and Sugiyama, 2007). Login to comment