ABCMdb

PMID: 18798335

Wang L, Dong H, Soroka CJ, Wei N, Boyer JL, Hochstrasser M
Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II.
Hepatology. 2008 Nov;48(5):1558-69., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg G238V, D482G, G982R, R1153C, and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Login to comment 5 ABCB11 p.Arg1153Cys Except for R1153C, the PFIC II mutants are degraded with varying half-lives. Login to comment 6 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. Login to comment 7 ABCB11 p.Asp482Gly The proteasome provides the major degradation pathway for the PFIC II mutants, whereas the lysosome also contributes to the degradation of D482G. Login to comment 10 ABCB11 p.Gly238Val Gene knockdown studies showed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, whereas HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (⌬Gly). Login to comment 11 ABCB11 p.Gly982Arg Furthermore, we present evidence that G982R weakly associates with various components of the ER quality control system. Login to comment 12 ABCB11 p.Arg1153Cys These data together demonstrate that the PFIC II mutants except R1153C and ⌬Gly are degraded by the ERAD pathway. Login to comment 31 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Gly238Val Inhibition of proteasomes also stabilized Bsep G238V, E297G, and D482G when examined in Madin-Darby canine kidney (MDCK) cells and human embryonic kidney (HEK) cells.8,10,13 These findings suggest that the proteasome plays a major role in the degradation of these BSEP mutants in PFIC II patients. Login to comment 48 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg The following missense mutants were studied in this work: G238V, D482G, G982R, R1153C, and R1286Q. Login to comment 82 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg The positions of G238V, D482G, G982R, R1153C, and R1286Q are indicated by star signs in a predicted topology model of rat Bsep. Login to comment 90 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val In contrast, the PFIC II mutants were mostly detected as core-glycosylated proteins, except G238V and D482G, for which a fraction of 190-kDa mature glycosylated form was also observed. Login to comment 95 ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg As can be seen, there is substantial colocalization between GFP-Bsep and calnexin in mutants that are exclusively core-glycosylated or non-glycosylated, that is, G982R, R1153C, R1286Q, and ⌬Gly. Login to comment 96 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val For G238V and D482G, which are partially core-glycosylated, there is a partial colocalization between GFP-Bsep and calnexin. Login to comment 99 ABCB11 p.Asp482Gly ABCB11 p.Gly982Arg Besides different glycosylation pattern, PFIC II mutants were also noticeably expressed at lower levels compared with the wt protein, with D482G, G982R, and ⌬Gly showing the lowest expression (Fig. 2A). Login to comment 107 ABCB11 p.Gly238Val In G238V, the core-glycosylated form disappears while the mature form is not significantly increased, indicating that the core-glycosylated form is degraded over the chase period. Login to comment 108 ABCB11 p.Asp482Gly For D482G, approximately 30% of the core-glycosylated Fig. 2. Login to comment 118 ABCB11 p.Asp482Gly protein was degraded, whereas a significant fraction was converted to the mature glycosylated form, indicating that this mutant is probably only weakly misfolded and a portion of correctly folded D482G can traffic through the secretory pathway. Login to comment 119 ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg G982R, R1286Q, and ⌬Gly were degraded over time as indicated by the decrease in the core-glycosylated form (the only form detected), whereas R1153C was relatively stable. Login to comment 122 ABCB11 p.Gly982Arg In contrast, G982R and R1286Q are degraded with a half-life of approximately 1 hour (Fig. 3C). Login to comment 123 ABCB11 p.Arg1153Cys R1153C is relatively stable. Login to comment 124 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val For G238V and D482G, respectively, only approximately 20% and 40% of the 140-kDa protein is converted to the 160-kDa protein, whereas approximately 50% of the 140-kDa protein is degraded over the 120-minute chase (Fig. 3D). Login to comment 125 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val These data suggest that the core-glycosylated G238V and D482G have a half-life of approximately 2 hours, which is consistent with the results from the cycloheximide chase studies, which used chase times up to 4 hours (Fig. 3A). Login to comment 126 ABCB11 p.Asp482Gly The higher conversion percentage for D482G is also consistent with the data in Fig. 3A. Login to comment 127 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val The Effect of Low Temperature and Chemical on G238V and D482G. Login to comment 128 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val The data in Fig. 3 suggest that a portion of D482G and G238V can be converted to the mature glycosylated form over time during biosynthesis. Login to comment 129 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly238Val We next asked whether conditions such as low temperature or chemical chaperones can stabilize D482G and G238V, because these conditions favor protein folding and have been shown to stabilize the misfolded mutant protein CFTR ⌬F508 during its biogenesis.19 Both incubation at low temperature (30°C) and addition of glycerol increases the peripheral, cell surface expression of GFP-tagged D482G and G238V in HEK cells (Fig. 4A). Login to comment 130 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val This is confirmed by a higher percentage of mature glycosylated form in D482G and G238V in HEK cells under these conditions (Fig. 4B). Login to comment 131 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val These data together confirm the notion that a fraction of correctly folded D482G and G238V can traffic through secretory pathway, and this fraction is increased under the conditions that favor protein folding. Login to comment 133 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg Because the proteasome and lysosome provide two of the major degradation mechanisms in the cell, we next examined the contribution of these two pathways to the stability of G238V, D482G, and G982R. Login to comment 147 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val Low temperature and glycerol stabilize G238V and D482G. Login to comment 148 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val (A) The HEK 293 cells were transfected with wt GFP-Bsep, G238V, and D482G. Login to comment 156 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg In contrast, treatment with MG132 significantly stabilized the 170-kDa core-glycosylated form of G982R, D482G, and G238V. Login to comment 158 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg In contrast, the combination of ammonium chloride, leupeptin, and pepstatin only moderately increases the mature glycosylated form of D482G, while not affecting the expression of G238V or G982R. Login to comment 161 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg (A) The HEK 293 cells were transfected with the wt GFP-Bsep, G238V, D482G, and G982R. Login to comment 168 ABCB11 p.Gly982Arg The HEK 293 cells were transfected with wt FLAG-Bsep (B) and G982R (C). Login to comment 173 ABCB11 p.Asp482Gly It is possible that D482G became prone to lysosome degradation when it traffics to a later stage of secretory pathway. Login to comment 174 ABCB11 p.Asp482Gly It is also worth noting that the 190-kDa mature glycosylated form of D482G also increases by the treatment of MG132 compared with the control condition. Login to comment 175 ABCB11 p.Asp482Gly This may reflect a situation that when the proteasome function is inhibited, the stabilized immature glycosylated D482G can traffic beyond the ER and is thus converted to the 190-kDa mature glycosylated form. Login to comment 176 ABCB11 p.Gly982Arg The stabilization of G982R by the proteasome inhibitor was confirmed by pulse-chase studies (Fig. 5C), whereas the expression of wt Bsep is little affected by MG132 (Fig. 5B). Login to comment 183 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg However, the increase in ubiquitination is moderate for wt Bsep, compared with the mutant proteins G238V, D482G, G982R, and ⌬Gly. Login to comment 193 ABCB11 p.Gly238Val To examine whether the ERAD E3s affect the expression of the Bsep mutants, we co-expressed the ERAD E3s HRD1, TEB4, Rma1, or CHIP with the wt Bsep, G238V, and ⌬Gly proteins. Login to comment 194 ABCB11 p.Gly238Val Increased levels of all the E3s resulted in decreased amounts of G238V and ⌬Gly compared with the mutant expressed alone (Fig. 7A). Login to comment 197 ABCB11 p.Gly238Val Enhanced ubiquitination of G238V was seen with the other ERAD E3s (data not shown). Login to comment 201 ABCB11 p.Gly238Val Reduction of TEB4 and Rma1 levels, but not those of CHIP, stabilized G238V. Login to comment 213 ABCB11 p.Gly238Val These data suggest that TEB4 and Rma1 may function as the E3s targeting G238V for degradation but do not do so for the wt Bsep and ⌬Gly proteins. Login to comment 218 ABCB11 p.Gly982Arg We transfected HEK cells with wt FLAG-Bsep, G982R and ⌬Gly, and isolated FLAG-Bsep by immunoprecipitation. Login to comment 221 ABCB11 p.Gly982Arg Weak binding was seen between the sugar-binding ER chaperone calnexin and wt Bsep and G982R but not with the nonglycosylated ⌬Gly mutant, as expected. Login to comment 222 ABCB11 p.Gly982Arg The ATPase p97, which extracts the retro-translocated proteins from the ER membrane,20 and a component of the retro-translocation complex HERP21,22 primarily associate with G982R as expected, which is a strong ERAD substrate. Login to comment 226 ABCB11 p.Gly238Val (A) The HEK 293 cells were transfected with wt GFP-Bsep, G238V, and ⌬Gly alone or co-transfected with the cDNAs encoding the ERAD E3s HRD1, TEB4, Rma1, and myc-CHIP. Login to comment 235 ABCB11 p.Gly238Val (A) The HEK 293 cells were transfected with wt GFP-Bsep, G238V, and ⌬Gly. Login to comment 242 ABCB11 p.Gly238Val (including digitonin, deoxyBigChap, NP-40, and Triton X-100) and solubilization conditions but could not detect any specific interaction between the endogenous HRD1, TEB4, and Rma1 E3s and Bsep mutant substrates such as G238V and ⌬Gly (data not shown). Login to comment 246 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg Taken together, the data from this study show that G238V, D482G, G982R, R1153C, R1286Q, and ⌬Gly mutations cause retention of Bsep in the ER to different extents. Login to comment 248 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly Although weakly expressed, a significant portion of D482G is expressed as mature glycosylated protein, which is consistent with previous reports showing that this mutant can be expressed at the cell surface and also retains bile salt transport function.8-10 These data together show that D482G is likely to be only a weakly misfolded mutant. Login to comment 249 ABCB11 p.Arg1153Cys Except for R1153C, the PFIC II mutants are short-lived, displaying distinct half-lives as measured by both cycloheximide chase and pulse-chase analyses. Login to comment 250 ABCB11 p.Gly982Arg Mutants such as G982R and R1286Q are degraded with a half-life of approximately 1 hour. Login to comment 251 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val For mutants D482G and G238V, only a portion of immature glycosylated protein can be converted to mature glycosylated protein over the chase period, whereas a large pool of immature glycosylated protein is degraded. Login to comment 252 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val This suggests that D482G and G238V are partly misfolded. Login to comment 253 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val This notion is supported by the data showing that conditions favoring protein folding, such as low temperature or the addition of glycerol, stabilize D482G and G238V and increase the fraction of the mature glycosylated form and cell surface expression (Fig. 4). Login to comment 254 ABCB11 p.Asp482Gly Proteasomes, rather than lysosomes, provide the major degradation pathway for the PFIC II mutants, whereas lysosome also moderately contributes to the degradation of D482G. Login to comment 255 ABCB11 p.Asp482Gly This may reflect a situation that D482G can traffic through the secretory pathway, and at different stages of the secretory pathway it can be sorted to proteasome or lysosome for degradation. Login to comment 256 ABCB11 p.Asp482Gly Kagawa et al.13 have recently also analyzed the degradation of D482G. Login to comment 257 ABCB11 p.Asp482Gly Comparable results were seen for the stabilization of D482G by proteasome inhibitor MG132 in MDCK cells. Login to comment 263 ABCB11 p.Asp482Gly ABCB11 p.Gly238Val ABCB11 p.Gly982Arg In addition, G238V, D482G, G982R, and ⌬Gly become relatively more ubiquitinated, compared with wt Bsep, when the function of the proteasome is inhibited, consistent with the notion that these mutant Bseps are misfolded and thus unstable. Login to comment 265 ABCB11 p.Gly982Arg The ERAD substrate G982R interacts with multiple components of the ER quality control system. Login to comment 266 ABCB11 p.Gly982Arg The HEK 293 cells were transfected with wt FLAG-Bsep, G982R, and ⌬Gly. Login to comment 274 ABCB11 p.Arg1153Cys R1153C is an interesting mutant, in that it is stable yet retained in the ER. Login to comment 275 ABCB11 p.Arg1153Cys The R1153C Bsep protein resembles two mutants of the yeast a-factor transporter Ste6p, Ste6- 13p, and Ste6-90p, which are retained in the ER and are hyperstable.24 In contrast, a large number of Ste6 mutants, such as Ste6-166p, are highly unstable and are degraded by the ERAD pathway. Login to comment 277 ABCB11 p.Arg1153Cys The R1153C mutant might be an example of this latter mechanism. Login to comment 278 ABCB11 p.Arg1153Cys ABCB11 p.Arg1153Cys R1153C most likely adopts a misfolded, bile salt transport-inactive conformation, because the mutant protein fails to transport taurocholate when expressed in Sf9 membrane vesicles.8 The misfolded R1153C is thus retained in the ER for continued folding attempts or sequestration instead of being degraded by the ERAD pathway. Login to comment 286 ABCB11 p.Gly238Val In contrast, siRNA-mediated knockdown of endogenous Rma1 and TEB4 stabilizes G238V but not ⌬Gly, whereas the knockdown of HRD1 levels significantly stabilizes ⌬Gly but not R1286Q. Login to comment 291 ABCB11 p.Gly982Arg Finally, we presented evidence that the ERAD substrates such as Bsep G982R weakly interact with components of the ER quality control system. Login to comment 294 ABCB11 p.Gly982Arg For example, p97 and HERP primarily associate with G982R, which is a strong ERAD substrate (Fig. 5). Login to comment 295 ABCB11 p.Gly982Arg ABCB11 p.Gly982Arg Although calnexin interacts with both wt Bsep and G982R, the ratio of calnexin bound to Bsep is likely to be higher for G982R, because its expression level is lower than wt Bsep. Login to comment 300 ABCB11 p.Asp482Gly This may be particularly pertinent to the rescue of those mutants that are partly misfolded but still functional, such as the D482G Bsep mutant. Login to comment