ABCMdb

PMID: 21184741

Sugiyama T, Shuto T, Suzuki S, Sato T, Koga T, Suico MA, Kusuhara H, Sugiyama Y, Cyr DM, Kai H
Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1.
Biochem Biophys Res Commun. 2011 Jan 21;404(3):853-8. Epub 2010 Dec 22., 2011-01-21 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Login to comment 28 ABCG2 p.Asn596Gln For example, not wild-type (WT) but N596Q variant of human BCRP, in which N-linked glycosylation was predicted not to occur at all, was susceptible to ER-associated degradation (ERAD) [9]. Login to comment 29 ABCG2 p.Gln141Lys ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Moreover, the certain single nucleotide polymorphism (SNP) variants of BCRP (Q141K, F208S and S441N), which protein expression was markedly low despite the functional expression of mRNA, were also degraded by ERAD [10,11]. Login to comment 31 ABCG2 p.Asn596Gln In the present study, we first screened ER-localized E3 ubiquitin ligases and their co-factor that functions in the regulation of WT and N596Q variant of BCRP expression and identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p [12], as a negative regulator for both glycosylated and non-glycosylated BCRP expression. Login to comment 32 ABCG2 p.Asn596Gln In addition, we demonstrated that the difference was observed between WT and N596Q variant of BCRP with respect to the mechanism underlying negative regulation of BCRP by Derlin-1. Login to comment 52 ABCG2 p.Asn596Gln Screening of ER-localized factors that function in the regulation of WT and N596Q variant of BCRP expression Although the posttranslational modification and ERAD of BCRP are becoming of great interest [8], there is only limited information on the specific factors that are critical for the regulation of BCRP expression. Login to comment 53 ABCG2 p.Asn596Gln In the present study, we focused on WT and N596Q variant of BCRP. Login to comment 57 ABCG2 p.Asn596Gln Whereas N596Q BCRP, in which Asn596 is substituted by Gln596, preferentially undergoes ERAD because of the lack of N-linked glycosylation although its PM targeting and transporter activity are less affected if it is trafficked from the ER to PM [9]. Login to comment 58 ABCG2 p.Asn596Gln We also checked the expression pattern of N596Q BCRP in HEK293 cells and confirmed the expression of non-glycosylated (Non-G) form of BCRP (Fig. 1A, lower pannel). Login to comment 62 ABCG2 p.Asn596Gln Notably, none of WT and dominant-negative mutant form of E3 ligases did not robustly affect the expression level and pattern of both WT and N596Q BCRP (Fig. 1B-E), while overexpression of Derlin-1 strongly reduced 81 kDa band (possibly C-G form) and increased 72 kDa band (possibly S-G form) of WT BCRP (Fig. 2F). Login to comment 63 ABCG2 p.Asn596Gln More interestingly, Derlin-1 overexpression strongly inhibited the expression level of N596Q BCRP (Fig. 2G). Login to comment 64 ABCG2 p.Asn596Gln Thus, these data suggest that Derlin-1, but not other known E3 ligases tested, may be the critical molecule to control the expression of both WT and N596Q BCRP proteins. Login to comment 78 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Derlin-1 facilitates ERAD of N596Q BCRP protein via reduction of its protein stability Since Derlin-1 overexpression suppressed expression of N596Q BCRP protein (Fig. 1G), we hypothesized that Derlin-1 could facilitate ERAD of N596Q BCRP protein. Login to comment 79 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Treatment with MG-132, a proteasome inhibitor [21], caused increased expression of N596Q BCRP protein, which is consistent with the previous study that showed that N596Q BCRP is relatively susceptible to ERAD (Fig. 3A) [9]. Login to comment 80 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Interestingly, knockdown of Derlin-1 significantly increased the expression of N596Q BCRP protein to a similar extent as MG-132 treatment (Fig. 3B and C), implying the possible involvement of Derlin-1 in the regulation of N596Q BCRP degradation. Login to comment 81 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Accordingly, significant reduction of N596Q BCRP expression was observed in the presence of Derlin-1 after 6 h CHX chase period (Fig. 3D and E), suggesting that both endogenous and exogenous Derlin-1 could reduce stability of N596Q protein and may contribute to ERAD of N596Q BCRP. Login to comment 84 ABCG2 p.Asn596Gln Based on our finding that Derlin-1 facilitates ERAD of non-glycosylated form of BCRP protein, N596Q BCRP, it is quite reasonable to think that Derlin-1 is also involved in tunicamycin-induced degradation of WT BCRP. Login to comment 86 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Further, pretreatment with MG132 inhibited the effect of tunicamycin-dependent change A - -+ - +- -BCRP 72 81 (kDa) S-G Non-G C-G Non-G EndoH PNGaseF WT BCRP N596Q BCRP - -+ - +- -BCRP 72 81 (kDa) EndoH PNGaseF B C -actin WT C42S Rma1 pcDNA 3.1 -Rma1 -BCRP 72 81 (kDa) WT C42S Rma1 pcDNA 3.1 WT BCRP N596Q BCRP S-G C-G Non-G actin Exo Rma1 Endo Rma1 D E F G -BCRP 72 -actin -Derlin-1 HA-Derlin-1 N596Q BCRP +- actin Non-G Exo Derlin-1 Endo Derlin-1 (kDa) -actin WT R2M gp78 pEF6 myc/his -myc -BCRP 72 81 (kDa) WT R2M gp78 WT BCRP N596Q BCRP S-G C-G Non-G actin gp78 pEF6 myc/his -actin WTC329S HRD1 pcDNA6 myc/his -myc -BCRP 72 81 (kDa) WT C329S HRD1 WT BCRP N596Q BCRP S-G C-G Non-G actin HRD1 pcDNA6 myc/his -actin WTH260Q CHIP pcDNA 3.1 -myc -BCRP 72 81 (kDa) WT H260Q CHIP WT BCRP N596Q BCRP S-G C-G Non-G actin CHIP pcDNA 3.1 HA-Derlin-1 WT BCRP +- -BCRP 72 81 -actin (kDa) actin S-G C-G Non-G -Derlin-1 Exo Derlin-1 Endo Derlin-1 Fig. 1. Login to comment 87 ABCG2 p.Asn596Gln Derlin-1 is the critical molecule to control the expression of both WT and N596Q BCRP proteins. Login to comment 88 ABCG2 p.Asn596Gln (A) HEK293 cells were transfected with WT or N596Q variant BCRP. Login to comment 90 ABCG2 p.Asn596Gln (B-E) WT or N596Q BCRP-transfected HEK293 cells were co-transfected with empty vectors or expression vectors that encode either WT or dominant-negative mutants of myc-tagged gp78 (B), Flag-tagged Rma1 (C), myc-tagged HRD1 (D) and myc-tagged CHIP (E). Login to comment 91 ABCG2 p.Asn596Gln (F-G) Empty vector pcDNA3.1 or HA-tagged Derlin-1 was co-transfected with WT (F) or N596Q (G) BCRP in HEK293 cells. Login to comment 104 ABCG2 p.Asn596Gln On the other hand, Derlin-1 is likely to be an ERAD mediator of N-linked glycosylation-deficient mutant N596Q BCRP and tunicamycin-induced non-glycosylated WT-BCRP proteins. Login to comment 127 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln A MG-132 N596Q BCRP +- -BCRP 72 -actin -Derlin-1 (kDa) actin Derlin-1 Non-G B si-Derlin-1 N596Q BCRP +- -BCRP 72 -actin -Derlin-1 (kDa) actin Derlin-1 Non-G C N596Q BCRP -actin 0 -Derlin-1 72 Non-G 3 6 pcDNA3.1 0 3 6 HA-Derlin-1 actin Exo Derlin-1 Endo Derlin-1 CHX chase (h) -BCRP (kDa) si-Derlin-1 N596Q BCRP +-D 0 25 50 75 100 125 150 175 0 3 6 (h) TotalBCRP%remaining pcDNA3.1 HA-Derlin-1 * CHX chase E RelativeQuantityof BCRPprotein 0 0.5 1 1.5 2 2.5 3 * Fig. 3. Login to comment 128 ABCG2 p.Asn596Gln Derlin-1 facilitates ERAD of N596Q BCRP protein via reduction of its protein stability. Login to comment 129 ABCG2 p.Asn596Gln (A) N596Q BCRP-transfected HEK293 cells were treated with 5 lM of MG132 for 24 h and lystaes were subjected to Westen blotting. Login to comment 130 ABCG2 p.Asn596Gln (B and C) N596Q BCRP-transfected HEK293 cells were co-transfected with 50 nM of control (À) or specific siRNA for Derlin-1 (+), followed by Western blotting (B). Login to comment 133 ABCG2 p.Asn596Gln (D, E) N596Q BCRP-transfected HEK293 cells were co-transfected with empty vector pcDNA3.1 or HA-tagged Derlin-1. Login to comment 135 ABCG2 p.Asn596Gln Non-G N596Q BCRP intensity was measured to express total BCRP amounts (E). Login to comment 137 ABCG2 p.Asn596Gln A -KDEL -Derlin-1 Derlin-1 72 81 S-G C-G Non-G -BCRP (kDa) 0 0.1 0.5 1 WT BCRP Tunicamycin -actin actin GRP94 GRP78 (µg/ml) -KDEL 72 81 S-G C-G -BCRP (kDa) -actin actin Non-G GRP94 GRP78 WT BCRP C - ++ - +- Tunicamycin MG-132 D -KDEL -Derlin-1 72 81 S-G C-G -BCRP (kDa) -actin actin Non-G Derlin-1 GRP94 GRP78 WT BCRP - ++ - +- Tunicamycin si-Derlin-1 Simply-glycosylated (S-G) BCRP ER Proteasome Degradation Non-glycosylated (Non-G) BCRP N596Q BCRP WT BCRP Golgi and plasma membrane Complex-glycosylated (C-G) BCRP -+ Derlin1 MG-132 - + Tunicamycin FE WT BCRP - ++ - +- Tunicamycin si-Derlin-1 * B RelativeQuantityofBCRP protein(C-G+Non-G) Tunicamycin (1 µg/ml) WT BCRP +- 0 0.2 0.4 0.6 0.8 1 1.2 * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 RelativeQuantityofBCRP protein(C-G+Non-G) Fig. 4. Login to comment 148 ABCG2 p.Asn596Gln (F) Schematic flow of Derlin1-dependent posttranslational regulation of glycosylated and non-glycosylated WT or N596Q BCRP. Login to comment 149 ABCG2 p.Asn596Gln Derlin-1 physically interacts with WT BCRP and reduces its maturation, and Derlin-1 initiates the efficient ERAD of non-glycosylated WT or N596Q BCRP. Login to comment 151 ABCG2 p.Asn596Gln MG-132 inhibits ERAD of Non-G WT-BCRP and N596Q BCRP. Login to comment 157 ABCG2 p.Asn596Gln Notably, overexpression of WT form of Rma1 and HRD1 but not dominant negative form of Rma1 (C42S) and HRD1 (C329S) seemed to slightly decrease N596Q BCRP expression (Fig. 1C and D), implicating that Derlin-1 may work with Rma1 and/or HRD1 during non-glycosylated BCRP degradation. Login to comment 162 ABCG2 p.Gln141Lys ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn These include naturally occurring SNPs variants of BCRP such as Q141K, F208S and S441N [10,11]. Login to comment 163 ABCG2 p.Asn596Gln ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu ABCG2 p.Arg383Ala Further, we could also assess the impact of Der- lin-1 on the expression of other BCRP variants such as R383A, G553L and G553E variants, which are also shown to be impaired N-linked glycosylation like N596Q BCRP [27,28]. Login to comment 165 ABCG2 p.Asn596Gln Overall, our data demonstrate that Derlin-1 interacts physically with WT BCRP and reduces its maturation, and Derlin-1 initiates the efficient ERAD of non-glycosylated WT and N596Q BCRP, indicating that Derlin-1 is the negative regulator for the expression of both glycosylated and non-glycosylated WT BCRP proteins. Login to comment