ABCG2 p.Cys592Gly
| Predicted by SNAP2: | A: D (66%), D: D (80%), E: D (80%), F: D (80%), G: D (80%), H: D (80%), I: D (71%), K: D (85%), L: D (75%), M: D (80%), N: D (80%), P: D (85%), Q: D (80%), R: D (85%), S: D (75%), T: D (59%), V: D (75%), W: D (80%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Human ABC transporter ABCG2 in xenobiotic protecti... Drug Metab Rev. 2006;38(3):371-91. Wakabayashi K, Tamura A, Saito H, Onishi Y, Ishikawa T
Human ABC transporter ABCG2 in xenobiotic protection and redox biology.
Drug Metab Rev. 2006;38(3):371-91., [PMID:16877258]
Abstract [show]
Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR/ABCP) is regarded as a member of the phase III system of xenobiotic metabolism. This efflux pump is suggested to be responsible for protecting the body from toxic xenobiotics and for removing toxic metabolites. The aim of this review article is to address new aspects of ABCG2 related to redox biology, namely the posttranslational modification (intra- and intermolecular disulfide bond formation) of ABCG2 protein and the transport of porphyrin and chlorophyll metabolites, as well as the high-speed screening and QSAR analysis method to evaluate ABCG2-drug interactions.
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No. Sentence Comment
65 The time course for the homodimer formation of the C592G/C608G variant after reductive treatment with dithiohreitol (Fig. 4B) was very similar to that observed for the ABCG2 WT.
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ABCG2 p.Cys592Gly 16877258:65:51
status: VERIFIED66 In the C592G/C608G variant, however, a significant part of the homodimer was less glycosylated, as suggested by the existence of an additional band with a smaller molecular size (Fig. 4A).
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ABCG2 p.Cys592Gly 16877258:66:7
status: VERIFIED78 Immunoblot detection of ABCG2 (WT) as well as of C603G and C592G/C608G variants expressed in Flp-In-293 cells (A).
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ABCG2 p.Cys592Gly 16877258:78:59
status: VERIFIED80 The effect of DTT treatment on C603G and C592G/C608G variants expressed in Flp-In-293 cells (B).
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ABCG2 p.Cys592Gly 16877258:80:41
status: VERIFIED92 These cysteine residues are considered to form an intramolecular disulfide bond, as illustrated in Fig. 4C. Although the C592G/C608G variant formed a homodimer, it exhibited low activity of ATP-dependent methotrexate (MTX) transport, and its cellular localization was remarkably altered.
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ABCG2 p.Cys592Gly 16877258:92:121
status: VERIFIED93 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced.
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ABCG2 p.Cys592Gly 16877258:93:12
status: VERIFIED94 This may explain the low level of drug resistance toward SN-38 or mitoxantrone in Flp-In-293/ ABCG2 (C592G/C608G) cells.
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ABCG2 p.Cys592Gly 16877258:94:101
status: VERIFIED95 Furthermore, immunoblotting of the C592G/C608G variant revealed the existence of a nonglycosylated form that was observed as an additional band with a lower molecular size (Fig. 4A).
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ABCG2 p.Cys592Gly 16877258:95:35
status: VERIFIED100 Substitution of either Cys592 or Cys608 to glycine resulted in a significant decrease in protein levels of ABCG2 in Flp-In-293 cells.
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ABCG2 p.Cys592Gly 16877258:100:23
status: VERIFIED101 Moreover, the protein level of the C592G/ C603G/C608G variant in Flp-In-293 cells was extremely low (Wakabayashi et al., 2006).
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ABCG2 p.Cys592Gly 16877258:101:35
status: VERIFIED[hide] Intramolecular disulfide bond is a critical check ... J Biol Chem. 2007 Sep 21;282(38):27841-6. Epub 2007 Aug 8. Wakabayashi K, Nakagawa H, Tamura A, Koshiba S, Hoshijima K, Komada M, Ishikawa T
Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein.
J Biol Chem. 2007 Sep 21;282(38):27841-6. Epub 2007 Aug 8., 2007-09-21 [PMID:17686774]
Abstract [show]
Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A(1), which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.
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No. Sentence Comment
8 On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A1, which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment.
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ABCG2 p.Cys592Gly 17686774:8:171
status: VERIFIED26 When both Cys-592 and Cys-608 were substituted to Gly, the resulting C592G/C608G variant could form a homodimer, but its expression level was remarkably reduced.
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ABCG2 p.Cys592Gly 17686774:26:69
status: VERIFIED35 38, pp. 27841-27846, September 21, 2007 (c) 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cys Role in ABCG2 Protein Stability Furthermore, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was not detectable (10).
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ABCG2 p.Cys592Gly 17686774:35:196
status: VERIFIED40 EXPERIMENTAL PROCEDURES Cell Culture-Flp-In-293 cells expressing ABCG2 WT or Cys-to-Gly variants (C592G and C608G) were established and cultured as described previously (10).
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ABCG2 p.Cys592Gly 17686774:40:98
status: VERIFIED58 RESULTS AND DISCUSSION Protein Expression Levels of ABCG2 WT, C592G, and C608G in Flp-In-293 Cells-We investigated the role of disulfide bond formation in the protein stability and cellular localization of ABCG2.
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ABCG2 p.Cys592Gly 17686774:58:62
status: VERIFIED64 B, expression of ABCG2 WT, C592G, and C608G in Flp-In-293 cells at the transcription and protein level.
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ABCG2 p.Cys592Gly 17686774:64:27
status: VERIFIED65 The transcription level was analyzed by reverse transcription-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G.
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ABCG2 p.Cys592Gly 17686774:65:134
status: VERIFIED68 DataareexpressedasmeanvaluesϮS.D.intriplicateexperiments.C,enhancementoftheproteinlevelofABCG2variantsbyMG132treatment.Flp-In-293cells expressing ABCG2 WT, C592G, or C608G were incubated with 2 M MG132 for 0, 24, and 48 h. ABCG2 protein levels were analyzed by immunoblotting after PNGase F treatment.
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ABCG2 p.Cys592Gly 17686774:68:162
status: VERIFIED79 A, immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G.
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ABCG2 p.Cys592Gly 17686774:79:72
status: VERIFIED81 Horizontal bars correspond to 20 m. B, effect of MG132 on ubiquitination of ABCG2 proteins.Afterincubationwithorwithout2M MG132for24h,celllysatesampleswerepreparedfromFlp-In-293cellsexpressingABCG2WT,C592G,orC608G.
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ABCG2 p.Cys592Gly 17686774:81:216
status: VERIFIED83 In the present study, we prepared two ABCG2 variants, C592G and C608G, in which Cys-592 and Cys-608 in the extracellular loop were substituted to Gly, respectively (Fig. 1A).
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ABCG2 p.Cys592Gly 17686774:83:54
status: VERIFIED85 Accordingly, as demonstrated in Fig. 1B, ABCG2 mRNA levels detected by reverse transcription-PCR were almost equal among ABCG2 WT, C592G, and C608G.
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ABCG2 p.Cys592Gly 17686774:85:131
status: VERIFIED86 However, substitution of either Cys-592 or Cys-608 to glycine resulted in a significant decrease in protein levels of ABCG2 in Flp-In-293 cells (Fig. 1B).
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ABCG2 p.Cys592Gly 17686774:86:32
status: VERIFIED87 Both C592G and C608G variants formed homodimers (data not shown; see Fig. 8 in Ref. 10).
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ABCG2 p.Cys592Gly 17686774:87:5
status: VERIFIED88 Immunoblotting without PNGase F treatment revealed two faint bands for each of the C592G and C608G variants, and these bands corresponded to glycosylated (MW ϭ 81,000) and non-glycosylated (MW ϭ 63,000) forms (Fig. 1B).
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ABCG2 p.Cys592Gly 17686774:88:83
status: VERIFIED90 After PNGase F treatment, one single band (MW ϭ 63,000) was detected not only for ABCG2 WT but also for the C592G and C608G variants (Fig. 1B).
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ABCG2 p.Cys592Gly 17686774:90:114
status: VERIFIED91 It is suggested that N-linked glycosylation was formed in the C592G and C608G variants as well as the WT in Flp-In-293 cells.
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ABCG2 p.Cys592Gly 17686774:91:62
status: VERIFIED92 Effect of MG132 or Bafilomycin A1 on the Protein Levels of ABCG2 WT, C592G, and C608G-As shown in Fig. 1C, the protein levels of those ABCG2 variants were remarkably enhanced by the treatment with the proteasome inhibitor MG132.
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ABCG2 p.Cys592Gly 17686774:92:69
status: VERIFIED94 The increases in protein levels of the ABCG2 C592G and C608G variants occurred in a time-dependent manner during the MG132 treatment.
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ABCG2 p.Cys592Gly 17686774:94:45
status: VERIFIED97 On the other hand, as depicted in Fig. 1D, the protein level of ABCG2 WT increased more than 5-fold when cells were treated with bafilomycin A1, which inhibits lysosomal degradation, whereas the C592G or C608G variants were little affected by the same treatment.
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ABCG2 p.Cys592Gly 17686774:97:195
status: VERIFIED99 Effect of MG132 on the Cellular Localization and Function of ABCG2 WT, C592G, and C608G-It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization and functional activity of the ABCG2 C592G and C608G variants.
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ABCG2 p.Cys592Gly 17686774:99:71
status: VERIFIEDX
ABCG2 p.Cys592Gly 17686774:99:255
status: VERIFIED100 Fig. 2A depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G that were incubated with or without 2 M MG132 for 24 h. ABCG2 proteins were probed with theBXP-21antibodyandthenlabeledwithgreenfluorescencedye (Alexa Fluor 488), whereas DNA in the nuclei was stained with propidium iodide (red fluorescence).
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ABCG2 p.Cys592Gly 17686774:100:87
status: VERIFIED102 A remarkable difference was observed after the MG132 treatment, however, in terms of the cellular localization of the C592G or C608G variant proteins.
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ABCG2 p.Cys592Gly 17686774:102:118
status: VERIFIED103 Without MG132 treatment, immunofluorescence of the C592G orC608Gvariantswasextremelyweakattheplasmamembraneof Flp-In-293 cells (Fig. 2A).
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ABCG2 p.Cys592Gly 17686774:103:51
status: VERIFIED104 Rather, those variant proteins were detected in intracellular compartments, suggesting that the sorting of C592G or C608G variants to the plasma membrane domain was impaired.
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ABCG2 p.Cys592Gly 17686774:104:107
status: VERIFIED105 It is noteworthy, however, that the plasma membrane localizations of the C592G and C608G variants were clearly detected after the MG132 treatment (Fig. 2A).
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ABCG2 p.Cys592Gly 17686774:105:73
status: VERIFIED106 To investigate the effect of MG132 on the ubiquitinated state of ABCG2 proteins, the Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G were incubated in the presence or absence of 2 M MG132 for 24 h.
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ABCG2 p.Cys592Gly 17686774:106:123
status: VERIFIED107 As demonstrated in Fig. 2B, significant increases in the ubiquitinated forms of the C592G and C608G variant proteins were detected by immunoblotting with the anti-ubiquitin antibody after immunoprecipitation with the anti-ABCG2 antibody BXP-21 (upper panel).
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ABCG2 p.Cys592Gly 17686774:107:84
status: VERIFIED111 Based on these results, it is suggested that both C592G and C608G variants that were sorted to the plasma membrane domain after the MG132 treatment were functionally active to extrude SN-38 out of the cells.
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ABCG2 p.Cys592Gly 17686774:111:50
status: VERIFIED117 C, drug resistance profiles of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G.
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ABCG2 p.Cys592Gly 17686774:117:69
status: VERIFIED122 D, schematic illustration for plausible pathways for protein processing and degradation of ABCG2 WT and Cys-to-Gly mutated variants (C592G and C608G).
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ABCG2 p.Cys592Gly 17686774:122:133
status: VERIFIED136 Misfolded proteins (e.g. ABCG2 C592G and C608G variants) may be removed from the ER by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system (24).
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ABCG2 p.Cys592Gly 17686774:136:31
status: VERIFIED142 In addition, we anticipate that some part of ubiquitinated ABCG2 C592G and C608G variants can undergo deubiquitination and subsequently be sorted to the plasma membrane domain (Fig. 2D) since these variants were markedly localized at the plasma membrane domain after MG132 treatments (Fig. 2A).
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ABCG2 p.Cys592Gly 17686774:142:65
status: VERIFIED[hide] Human ABC transporters ABCG2 (BCRP) and ABCG4. Xenobiotica. 2008 Jul;38(7-8):863-88. Koshiba S, An R, Saito H, Wakabayashi K, Tamura A, Ishikawa T
Human ABC transporters ABCG2 (BCRP) and ABCG4.
Xenobiotica. 2008 Jul;38(7-8):863-88., [PMID:18668433]
Abstract [show]
1. The human ABC transporter ABCG2 is regarded as a member of the phase III system for xenobiotic metabolism, and it has been suggested that this efflux pump is responsible for protecting the body from toxic xenobiotics and for removing metabolites. 2. This review paper will address the new aspects of ABCG2 in terms of post-translational modifications (i.e., disulfide bond formation, ubiquitination, and endoplasmic reticulum-associated degradation) of ABCG2 protein, high-speed screening, and quantitative structure-activity relationship (QSAR) analysis to evaluate ABCG2-drug interactions, and genetic polymorphisms potentially associated with photosensitivity. 3. In addition, new aspects of human ABCG4 and mouse Abcg4 are presented with respect to their molecular properties and potential physiological roles. Considering a high sequence similarity between ABCG1 and ABCG4, both Abcg4 and ABCG4 may be involved in the transport of cholesterol from neurons and astrocytes. Furthermore, high expression of the mouse Abcg4 protein in the testis implicates its involvement in transport of certain sex hormones.
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No. Sentence Comment
63 Substitution of either Cys592 or Cys608 with glycine resulted in a significant decrease in protein expression levels of ABCG2 in Flp-In-293 cells, while mRNA levels were almost equal (Figure 3a).
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ABCG2 p.Cys592Gly 18668433:63:23
status: VERIFIED67 Indeed, treatment with the proteasome inhibitor MG132 substantially enhanced the protein levels of those ABCG2 variants (C592G and C608G) in Flp-In-293 cells with a concomitant increase in ubiquitinated forms of those variant proteins detected by immunoprecipitation (Wakabayashi et al. 2007).
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ABCG2 p.Cys592Gly 18668433:67:121
status: VERIFIED70 On the other hand, the protein level of ABCG2 WT increased more than four-fold when cells were treated with bafilomycin A1, which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment (Wakabayashi et al. 2007).
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ABCG2 p.Cys592Gly 18668433:70:174
status: VERIFIED80 (a) Expression of ABCG2 WT, C592G, and C608G in Flp-In-293 cells at the transcription and protein level.
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ABCG2 p.Cys592Gly 18668433:80:28
status: VERIFIED81 The transcription level was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G.
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ABCG2 p.Cys592Gly 18668433:81:165
status: VERIFIED86 Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G were incubated with 2 mM MG132 for zero, 24, and 48 h. ABCG2 protein levels were analysed by immunoblotting after PNGase F treatment.
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ABCG2 p.Cys592Gly 18668433:86:38
status: VERIFIED99 If these misfolded proteins are not degraded in proteasomes (e.g., MG132 inhibition), they are transported in a microtubule-organizing centre together with ubiquitin and ER chaperones Figure 4. Schematic illustration for plausible pathways for protein processing and degradation of ABCG2 WT and Cys-to-Gly mutated variants (C592G and C608G).
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ABCG2 p.Cys592Gly 18668433:99:324
status: VERIFIED[hide] Quality control of human ABCG2 protein in the endo... Adv Drug Deliv Rev. 2009 Jan 31;61(1):66-72. Epub 2008 Dec 11. Wakabayashi-Nakao K, Tamura A, Furukawa T, Nakagawa H, Ishikawa T
Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):66-72. Epub 2008 Dec 11., 2009-01-31 [PMID:19111842]
Abstract [show]
Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR/ABCP) is a plasma membrane protein carrying intra- and inter-molecular disulfide bonds and an N-linked glycan. Both disulfide bond formation and N-glycosylation are critical check points determining the stability and degradation fate of ABCG2 protein in the endoplasmic reticulum (ER). Misfolded ABCG2 protein without those post-translational modifications is removed from the ER by retrotranslocation to the cytosol compartment, ubiquitination by ubiquitin ligase, and finally degradation by proteasomes. Certain non-synonymous SNP variants of ABCG2 undergo such ER-associated degradation (ERAD).
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No. Sentence Comment
891 Substitution of either Cys592 or Cys608 to glycine resulted in a significant decrease in protein levels of ABCG2 in Flp-In-293 cells, while the mRNA levels of ABCG2 WT and those variants were almost equal because of the Flp-mediated integration of those cDNAs [7].
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ABCG2 p.Cys592Gly 19111842:891:23
status: NEW892 Moreover, the protein level of the C592G/C603G/ C608G variant in Flp-In-293 cells was extremely low [7].
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ABCG2 p.Cys592Gly 19111842:892:35
status: NEW893 Although the C592G/C608G variant formed a homodimer, it exhibited lower ATP-dependent MTX transport activity and its cellular localization was remarkably altered.
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ABCG2 p.Cys592Gly 19111842:893:13
status: NEW894 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced.
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ABCG2 p.Cys592Gly 19111842:894:12
status: NEW895 Furthermore, immunoblotting of the C592G/C608G variant revealed the existence of a non-glycosylated form that was observed as an additional band with a lower molecular weight [7,8].
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ABCG2 p.Cys592Gly 19111842:895:35
status: NEW898 As a misfolded protein, the C592G/C608G variant appears to be removed from the ER by retrotranslocation to the cytosol and then degradation by the ubiquitin-proteasome system [8].
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ABCG2 p.Cys592Gly 19111842:898:28
status: NEW[hide] Human ABC transporter ABCG2 in cancer chemotherapy... J Exp Ther Oncol. 2009;8(1):5-24. Ishikawa T, Nakagawa H
Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics.
J Exp Ther Oncol. 2009;8(1):5-24., [PMID:19827267]
Abstract [show]
The ability of cancer cells to acquire resistance to multiple anticancer agents, termed multidrug resistance, is often mediated by overexpression of ATP-binding cassette (ABC) transporters that remove drugs out of the cell against a concentration gradient. ABCG2, or breast cancer resistance protein (BCRP), is an ABC transporter that has been the subject of intense study since its discovery a decade ago. While ABCG2 overexpression has been demonstrated in cancer cells after in vitro drug treatment, endogenous ABCG2 expression in certain cancers is considered as a reflection of the differentiated phenotype of the cell of origin and likely contributes to intrinsic drug resistance. Notably, ABCG2 is often expressed in stem cell populations, where it plays a critical role in cellular protection. ABCG2 exhibits a broad range of substrate specificity. New technologies of high-speed screening and quantitative structure-activity-relationship (QSAR) analysis have been developed to analyze the interactions of drugs with ABCG2. As ABCG2 reportedly transports porphyrins, its contribution to photodynamic therapy of human cancer is also implicated. Protein expression levels of ABCG2 in cancer cells are regulated by both transcriptional activation and protein degradation. The ABCG2 protein undergoes endosomal and/or ubiquitin-mediated proteasomal degradations. Furthermore, genetic polymorphisms in the ABCG2 gene are important factors in cancer chemotherapy to circumvent adverse effects and/or to enhance the efficacy of anticancer drugs. The present review article addresses recent advances in molecular pharmacology and pharmacogenomics of ABCG2 and provides novelideas to improve cancer chemotherapy.
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No. Sentence Comment
190 Although the C592G/C608G variant formed a homodimer, it exhibited lower ATP-dependent MTX transport activity and its cellular localization was remarkably altered.
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ABCG2 p.Cys592Gly 19827267:190:13
status: VERIFIED191 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced.
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ABCG2 p.Cys592Gly 19827267:191:12
status: VERIFIED192 Furthermore, immunoblotting of the C592G/C608G variant revealed the existence of a non-glycosylated form that was observed as an additional band with a lower molecular weight (Wakabayashi et al., 2006a; Wakabayashi et al. 2007).
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ABCG2 p.Cys592Gly 19827267:192:35
status: VERIFIED194 As a misfolded protein, the C592G/C608G variant appears to be removed from the endoplasmic reticulum (ER) by retrotranslocation to the cytosol and then degradation by the ubiquitin-proteasome system (Wakabayashi et al., 2007).
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ABCG2 p.Cys592Gly 19827267:194:28
status: VERIFIED[hide] Receptor for activated C-kinase 1 regulates the ce... Drug Metab Dispos. 2010 Dec;38(12):2320-8. Epub 2010 Sep 21. Ikebuchi Y, Ito K, Takada T, Anzai N, Kanai Y, Suzuki H
Receptor for activated C-kinase 1 regulates the cell surface expression and function of ATP binding cassette G2.
Drug Metab Dispos. 2010 Dec;38(12):2320-8. Epub 2010 Sep 21., [PMID:20858845]
Abstract [show]
In a previous report, we identified the receptor for activated C-kinase 1 (RACK1) as a positive regulator of the cellular localization and expression of ATP-binding cassette B4, a phosphatidylcholine translocator expressed on the bile canalicular membrane. In the present study, we focused on the role of RACK1 on ATP-binding cassette G2 (ABCG2), which is responsible for the cellular extrusion of compounds including antitumor drugs. Protein expression of ABCG2 was up-regulated by RACK1 overexpression, although mRNA expression of ABCG2 was not dependent on RACK1. The effect of RACK1 on the expression of ABCG2 on the cell surface was confirmed by the uptake of [(3)H]estrone sulfate, an ABCG2 substrate, into isolated membrane vesicles. The expression of RACK1 affected cellular resistance to mitoxantrone, an anticancer drug excreted by ABCG2, and this effect of RACK1 was abolished in the presence of fumitremorgin C, a selective ABCG2 inhibitor. These results suggest that RACK1 has functional significance as a regulatory cofactor of ABCG2 and is indispensable for the cell surface expression and excretion function of ABCG2. The precise mechanism for RACK1-dependent expression of ABCG2 remains to be clarified, because the results of N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and chloroquine treatment and those of metabolic labeling experiments did not give us clear evidence whether the reduction of ABCG2 expression in RACK1-knocked down cells may be caused by the suppression of ABCG2 protein synthesis or by acceleration of its degradation.
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No. Sentence Comment
279 Another study showed that the expression level of the ABCG2 C592G/C608G mutant, which lacks the intramolecular disulfide bond, was increased by MG132 treatment, whereas that of the wild type was not affected (Wakabayashi et al., 2007).
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ABCG2 p.Cys592Gly 20858845:279:60
status: VERIFIED[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]
Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.
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No. Sentence Comment
92 In fact, substitution of either Cys592 or Cys608 to Gly resulted in a marked decrease in protein levels of ABCG2 in the plasma membrane of Flp-In-293 cells in vitro.77 Moreover, the C592G/ C603G/C608G variant, in which all of the three Cys residues were substituted to Gly, was expressed at an extremely low level.73 The reduced protein levels could not be ascribed to any instability of mRNA encoding those variants because the mRNA levels of ABCG2 WT and those variants were kept at equal levels by Flp-mediated chromosomal integration of one single copy of ABCG2 complementary DNA (cDNA).73 It is important to note that the C592G/C608G variant formed a homodimer, however its cellular localization was remarkably altered.
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ABCG2 p.Cys592Gly 21567408:92:32
status: NEWX
ABCG2 p.Cys592Gly 21567408:92:182
status: NEWX
ABCG2 p.Cys592Gly 21567408:92:627
status: NEW93 Indeed, the C592G/ C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced.
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ABCG2 p.Cys592Gly 21567408:93:12
status: NEW94 Interestingly, the ABCG2 variant formed aggresomes,83 when proteasomal degradation was inhibited by MG132 treatments in Flp-In-293 cells.77,84 Furthermore, immunoblotting of the C592G/C608G variant revealed the formation of a nonglycosylated ABCG2 protein.73,77 Those observations strongly suggest that the intramolecular disulfide bond formation between Cys592 and Cys608 is a prerequisite not only for cellular localization but also for N-linked glycosylation of the ABCG2 protein. As Asn596 is an N-linked glycosylation site in the ABCG2 protein, the formation of the intramolecular disulfide bond between Cys592 and Cys608 may facilitate the access of oligosaccharyl transferase to Asn596 for N-linked glycosylation (Fig. 4).
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ABCG2 p.Cys592Gly 21567408:94:178
status: NEW[hide] Targeted degradation of ABC transporters in health... J Bioenerg Biomembr. 2007 Dec;39(5-6):489-97. Nikles D, Tampe R
Targeted degradation of ABC transporters in health and disease.
J Bioenerg Biomembr. 2007 Dec;39(5-6):489-97., [PMID:17972020]
Abstract [show]
ATP binding cassette (ABC) transporters comprise an extended protein family involved in the transport of a broad spectrum of solutes across membranes. They consist of a common architecture including two ATP-binding domains converting chemical energy into conformational changes and two transmembrane domains facilitating transport via alternating access. This review focuses on the biogenesis, and more precisely, on the degradation of mammalian ABC transporters in the endoplasmic reticulum (ER). We enlighten the ER-associated degradation pathway in the context of misfolded, misassembled or tightly regulated ABC transporters with a closer view on the cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP), which plays an essential role in the adaptive immunity. Three rather different scenarios affecting the stability and degradation of ABC transporters are discussed: (1) misfolded domains caused by a lack of proper intra- and intermolecular contacts within the ABC transporters, (2) deficient assembly with auxiliary factors, and (3) arrest and accumulation of an intermediate or 'dead-end' state in the transport cycle, which is prone to be recognized by the ER-associated degradation machinery.
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92 Table 1 Degradation of ABC transporters ABC transporter Organism Initiation of degradation Ref. CFTR/ABCC7 (wt and ΔF508) Human Misfolding Jensen et al. (1995), Ward et al. (1995) SUR1/ABCC8 (wt and mutants) Human Lack of K(ATP) channel misfolding Crane and Aguilar-Bryan (2004); Yan et al. (2005) ABCG2 (C592G or C608G) Human Lack of intramolecular disulfide bond Wakabayashi et al. (2007) ALDP/ABCD1 (several mutants) Human Mutations in the NBD Takahashi et al. (2007) TAP2/ABCB3 Human Lack of TAP1 de la Salle et al. (1999), Heintke et al. (2003), Karttunen et al. (2001) TAP1/2/ABCB2/3 Human Lack of tapasin Garbi et al. (2003), Lehner et al. (1998), Papadopoulos and Momburg (2007) TAP1/2/ABCB2/3 Human Viral inhibitors UL49.5 and mK3 Boname et al. (2004), Koppers-Lalic et al. (2005), Lybarger et al. (2003), Wang et al. (2007) Pdr5 (ΔC-term and L183P) Yeast Misfolded NBD de Thozee et al. (2007) Yor1p (ΔF670) Yeast Space change in NBD Pagant et al. (2007) Instability and degradation of TAP2 in TAP1 deficiency (Bare Lymphocyte Syndrome, BLS) Molecules and disease-associated states that interfere with the stability of TAP are reconciled in this and the next chapter to reveal an overall understanding of TAP biogenesis.
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ABCG2 p.Cys592Gly 17972020:92:311
status: NEW