ABCMdb

ABCG2 p.Cys592Gly

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Publications

PMID: 16877258 [PubMed] Wakabayashi K et al: "Human ABC transporter ABCG2 in xenobiotic protection and redox biology."

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. Login to comment
66 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). Login to comment
78 Immunoblot detection of ABCG2 (WT) as well as of C603G and C592G/C608G variants expressed in Flp-In-293 cells (A). Login to comment
80 The effect of DTT treatment on C603G and C592G/C608G variants expressed in Flp-In-293 cells (B). Login to comment
92 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. Login to comment
93 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced. Login to comment
94 This may explain the low level of drug resistance toward SN-38 or mitoxantrone in Flp-In-293/ ABCG2 (C592G/C608G) cells. Login to comment
95 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). Login to comment
100 Substitution of either Cys592 or Cys608 to glycine resulted in a significant decrease in protein levels of ABCG2 in Flp-In-293 cells. Login to comment
101 Moreover, the protein level of the C592G/ C603G/C608G variant in Flp-In-293 cells was extremely low (Wakabayashi et al., 2006). Login to comment

PMID: 17686774 [PubMed] Wakabayashi K et al: "Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein."

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. Login to comment
26 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. Login to comment
35 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). Login to comment
40 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). Login to comment
58 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. Login to comment
64 B, expression of ABCG2 WT, C592G, and C608G in Flp-In-293 cells at the transcription and protein level. Login to comment
65 The transcription level was analyzed by reverse transcription-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment
68 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. Login to comment
79 A, immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment
81 Horizontal bars correspond to 20 ␮m. B, effect of MG132 on ubiquitination of ABCG2 proteins.Afterincubationwithorwithout2␮M MG132for24h,celllysatesampleswerepreparedfromFlp-In-293cellsexpressingABCG2WT,C592G,orC608G. Login to comment
83 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). Login to comment
85 Accordingly, as demonstrated in Fig. 1B, ABCG2 mRNA levels detected by reverse transcription-PCR were almost equal among ABCG2 WT, C592G, and C608G. Login to comment
86 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). Login to comment
87 Both C592G and C608G variants formed homodimers (data not shown; see Fig. 8 in Ref. 10). Login to comment
88 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). Login to comment
90 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). Login to comment
91 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. Login to comment
92 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. Login to comment
94 The increases in protein levels of the ABCG2 C592G and C608G variants occurred in a time-dependent manner during the MG132 treatment. Login to comment
97 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. Login to comment
99 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. Login to comment
100 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). Login to comment
102 A remarkable difference was observed after the MG132 treatment, however, in terms of the cellular localization of the C592G or C608G variant proteins. Login to comment
103 Without MG132 treatment, immunofluorescence of the C592G orC608Gvariantswasextremelyweakattheplasmamembraneof Flp-In-293 cells (Fig. 2A). Login to comment
104 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. Login to comment
105 It is noteworthy, however, that the plasma membrane localizations of the C592G and C608G variants were clearly detected after the MG132 treatment (Fig. 2A). Login to comment
106 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. Login to comment
107 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). Login to comment
111 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. Login to comment
117 C, drug resistance profiles of Flp-In-293 cells expressing ABCG2 WT, C592G, or C608G. Login to comment
122 D, schematic illustration for plausible pathways for protein processing and degradation of ABCG2 WT and Cys-to-Gly mutated variants (C592G and C608G). Login to comment
136 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). Login to comment
142 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). Login to comment

PMID: 18668433 [PubMed] Koshiba S et al: "Human ABC transporters ABCG2 (BCRP) and ABCG4."

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). Login to comment
67 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). Login to comment
70 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). Login to comment
80 (a) Expression of ABCG2 WT, C592G, and C608G in Flp-In-293 cells at the transcription and protein level. Login to comment
81 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. Login to comment
86 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. Login to comment
99 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). Login to comment

PMID: 19111842 [PubMed] Wakabayashi-Nakao K et al: "Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation."

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]. Login to comment
892 Moreover, the protein level of the C592G/C603G/ C608G variant in Flp-In-293 cells was extremely low [7]. Login to comment
893 Although the C592G/C608G variant formed a homodimer, it exhibited lower ATP-dependent MTX transport activity and its cellular localization was remarkably altered. Login to comment
894 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced. Login to comment
895 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]. Login to comment
898 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]. Login to comment

PMID: 19827267 [PubMed] Ishikawa T et al: "Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics."

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. Login to comment
191 Indeed, the C592G/C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced. Login to comment
192 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). Login to comment
194 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). Login to comment

PMID: 20858845 [PubMed] Ikebuchi Y et al: "Receptor for activated C-kinase 1 regulates the cell surface expression and function of ATP binding cassette G2."

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). Login to comment

PMID: 21567408 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts."

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. Login to comment
93 Indeed, the C592G/ C608G variant was mainly localized in intracellular compartments, and its plasma membrane localization was significantly reduced. Login to comment
94 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). Login to comment

PMID: 17972020 [PubMed] Nikles D et al: "Targeted degradation of ABC transporters in health and disease."

No. Sentence Comment

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. Login to comment