ABCC1 p.Cys265Ser
Predicted by SNAP2: | A: N (78%), D: N (53%), E: N (61%), F: N (78%), G: N (57%), H: N (87%), I: N (78%), K: N (72%), L: N (87%), M: N (82%), N: N (78%), P: N (53%), Q: N (82%), R: N (72%), S: N (78%), T: N (82%), V: N (87%), W: N (72%), Y: N (87%), |
Predicted by PROVEAN: | A: N, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: D, Y: D, |
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[hide] Intracellular trafficking of MDR transporters and ... Curr Top Med Chem. 2009;9(2):197-208. Porcelli L, Lemos C, Peters GJ, Paradiso A, Azzariti A
Intracellular trafficking of MDR transporters and relevance of SNPs.
Curr Top Med Chem. 2009;9(2):197-208., [PMID:19200005]
Abstract [show]
Multi-drug resistance (MDR) frequently contributes to the failure of chemotherapeutic treatments in cancer patients. Mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Among them, the ATP-Binding Cassette (ABC) family of proteins plays a pivotal role. Processes of cellular distribution and subcellular localization of MDR-ABC proteins are not yet well explored and to enlighten these topics could be crucial to understand cellular drug uptake and retention. In this review, we analysed literature data concerning i) intracellular trafficking of MDR-ABC proteins (BCRP, P-gp and MRP1) and ii) mechanisms altering their cellular localization and trafficking. Moreover, we describe single nucleotide polymorphisms (SNP) that have been reported for some multidrug resistance (MDR) transporters, such as BCRP and P-gp, emphasizing their ability to affect the expression, function and localization of the transporters, with implications on drug resistance phenotypes.
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No. Sentence Comment
241 In particular, the cell lines expressing the variants Cys43Ser-ABCC1, Cys 265Ala-ABCC1 and Cys265Ser-ABCC1 exhibited severely disrupted plasma membrane trafficking [116].
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ABCC1 p.Cys265Ser 19200005:241:91
status: NEW[hide] Functional and structural consequences of cysteine... Biochemistry. 2003 May 13;42(18):5214-24. Leslie EM, Letourneau IJ, Deeley RG, Cole SP
Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1).
Biochemistry. 2003 May 13;42(18):5214-24., 2003-05-13 [PMID:12731862]
Abstract [show]
The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.
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54 Mutagenesis was performed according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined and introduced or lost restriction sites are in italics) as follows: Cys43Ala (5'-G TGG GTG CCT GCT TTT TAC CTC TGG GCC-3'), Cys43Ser (5'-G TGG GTG CCT TCT TTT TAC CTC-3'), Cys49Ala (5'-C CTC TGG GCC GCA TTC CCC TTC TAC-3') (BsmI), Cys49Ser (5'-C CTC TGG GCC TCT TTC CCC TTC-3'), Cys85Ala (5'-G TGG ATC GTC GCG TGG GCA GAC C-3') (BstUI), Cys85Ser (5'-G TGG ATC GTC AGC TGG GCA GAC C-3'), Cys148Ala (5'-GTA GCC CTA GTG GCT GCC CTA GCC-3') (BglI), Cys148Ser (5'-GTA GCC CTA GTG TCT GCC CTA GCC-3'), Cys190Ala (5'-C GTC TTG TCC GCA TTC TCA GAT CGC-3') (BsmI), Cys190Ser (5'-C GTC TTG TCC TCT TTC TCA GAT CG-3'), Cys208Ala (5'-C CCT AAT CCC GCG CCA GAG TCC AG-3') (BstUI), Cys208Ser (5'-C CCT AAT CCC AGC CCA GAG TCC-3'), Cys265Ala (5'-GTA AAG AAC TGG AAG AAG GAA GCC GCG AAG ACT AGG AAG CAG-3') (BpiI), and Cys265Ser (5'- GG AAG AAG GAA TCC GCC AAG ACT AG-3') (BsmI).
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ABCC1 p.Cys265Ser 12731862:54:958
status: NEW109 Thus, the cell lines expressing the TM1 mutant Cys43Ser-MRP1 (Figure 3D) and the CL3 mutants Cys265Ala-MRP1 (Figure 3O) and Cys265Ser-MRP1 (Figure 3P) exhibited severely disrupted plasma membrane trafficking.
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ABCC1 p.Cys265Ser 12731862:109:124
status: NEW111 Three of the mutant cell lines with impaired plasma membrane trafficking of MRP1 exhibited filament-like staining (Cys43Ala, Cys43Ser, and Cys49Ser) (Figure 3C,D,F) while the cell lines expressing Cys265Ala and Cys265Ser MRP1 showed a stippled-like staining pattern.
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ABCC1 p.Cys265Ser 12731862:111:211
status: NEW112 Finally, Cys265Ser expressing cells had a unique staining pattern, which was stippled and resembled vacuolar staining.
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ABCC1 p.Cys265Ser 12731862:112:9
status: NEW130 Table 1: Detection of Tryptic Fragments N1 and N2 of MRP1 in Membranes Prepared from HeLa Cells Stably Expressing CysfAla and CysfSer MRP1 Mutantsa trypsin:protein ratio (w:w)transfected HeLa cell line N1 detected N2 detected WT-MRP1 1:10 000 1:1000 C43A-MRP1 1:100 1:100 C43S-MRP1 1:10 000 1:1000 C49A-MRP1 1:250 1:100 C49S-MRP1 1:10 000 1:500 C85A-MRP1 1:10 000 1:1000 C85S-MRP1 1:1000 1:250 C148A-MRP1 1:250 1:250 C148S-MRP1 1:1000 1:500 C190A-MRP1 1:1000 1:1000 C190S-MRP1 1:1000 1:250 C208A-MRP1 1:10 000 1:250 C208S-MRP1 1:10 000 1:500 C265A-MRP1 1:250 1:10 C265S-MRP1 1:1000 1:250 a The data shown represent a summary of the limited trypsin digests shown in Figures 4 and 5.
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ABCC1 p.Cys265Ser 12731862:130:564
status: NEW132 Cys49Ser, Cys190Ala, Cys208Ala, and Cys208Ser also closely matched those of WT-MRP1 except that the N1 fragment did not appear until a trypsin:protein ratio of 1:1000 for Cys190Ala-MRP1 and the N2 fragments of Cys49Ser-MRP1, Cys208Ala-MRP1, and Cys208Ser-MRP1 did not appear until trypsin:protein ratios of 1:500, 1:250, and 1:500, respectively. The remaining CysfSer MRP1 mutants (Cys85Ser, Cys148Ser, Cys190Ser, and Cys265Ser) were more resistant to trypsinolysis than WT-MRP1 but less resistant than their respective Ala partners.
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ABCC1 p.Cys265Ser 12731862:132:418
status: NEW133 For all four of these CysfSer mutants, the N1 tryptic fragment appeared at a trypsin:protein ratio of 1:1000 while the N2 fragment appeared at ratios of 1:500 for Cys148Ser and 1:250 for Cys85Ser, Cys190Ser, and Cys265Ser.
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ABCC1 p.Cys265Ser 12731862:133:212
status: NEW151 In contrast, unlike Cys265Ala-MRP1, which showed increased activity, Cys265Ser-MRP1 exhibited LTC4 uptake activity similar to WT-MRP1.
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ABCC1 p.Cys265Ser 12731862:151:69
status: NEW156 Consistent with their Ala-substituted counterparts, Cys49Ser-MRP1, Cys148Ser- MRP1, and Cys265Ser-MRP1 showed moderately reduced E217 G uptake (34-50%) while uptake by Cys43Ser-MRP1 was increased by 40% relative to WT-MRP1.
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ABCC1 p.Cys265Ser 12731862:156:88
status: NEW177 Thus, the arsenite resistance of cells expressing the TM1 Cys43Ser mutant was 2.5-fold lower than the cell line expressing WT-MRP1 whereas cells expressing Cys265Ser MRP1 were approximately 3-fold more resistant to this heavy metal oxyanion (Table 2).
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ABCC1 p.Cys265Ser 12731862:177:156
status: NEW178 Representative chemosensitivity assays illustrating the sodium arsenite resistance of cell lines expressing the Cys43Ser and Cys265Ser MRP1 mutants are shown in Figure 7.
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ABCC1 p.Cys265Ser 12731862:178:125
status: NEW179 The HeLa cell lines expressing Cys43Ser-MRP1 and Cys265Ser-MRP1 that showed changes in arsenite resistance were also tested for their sensitivity to the anticancer drugs vincristine and doxorubicin.
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ABCC1 p.Cys265Ser 12731862:179:49
status: NEW181 The Cys43Ala, Cys43Ser, Cys265Ala, and Cys265Ser MRP1 mutant expressing cell lines were 6.5-, 2.6-, 7.1-, and 5.1-fold resistant to doxorubicin, respectively, levels of resistance that did not differ significantly from the ~5-fold resistance observed with cells expressing WT-MRP1 (Figure 8A).
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ABCC1 p.Cys265Ser 12731862:181:39
status: NEW182 In contrast, the cell line expressing Cys43Ser-MRP1 was only 5-fold resistant to vincristine while the cell lines expressing Cys43Ala, Cys265Ala, and Cys265Ser-MRP1 were 21-, 19-, and 13-fold resistant, respectively, levels of resistance comparable to those observed in cells expressing WT-MRP1 (15-fold resistant) (Figure 8B).
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ABCC1 p.Cys265Ser 12731862:182:150
status: NEW192 Table 2: Sensitivity of Stably Transfected HeLa Cells Expressing Wild-Type and Cys-Substituted MRP1 to Sodium Arsenite and Potassium Antimony Tartrate relative resistancea transfected HeLa cell line Na+ arsenite K+ antimony tartrate WT-MRP1 3.6 ( 1.3 (1) (n ) 6) 2.0 ( 0.5 (1) (n ) 7) C43A-MRP1 4.3 ( 0.7 (1.2) (n ) 3) 2.0 ( 0.4 (1) (n ) 3) C43S-MRP1 1.4 ( 0.5 (0.4)b (n ) 6) 2.8 ( 1.1 (1.4) (n ) 4) C49A-MRP1 4.0 ( 1.6 (1.1) (n ) 5) 2.6 ( 0.7 (1.3) (n ) 4) C49S-MRP1 3.0 ( 1.5 (0.8) (n ) 4) 3.0 ( 0.6 (1.5) (n ) 4) C190A-MRP1 3.8 ( 0.4 (1) (n ) 4) 4.0 ( 1.2 (2) (n ) 3) C190S-MRP1 2.9 ( 0.4 (0.8) (n ) 4) 2.7 ( 0.4 (1.4) (n ) 3) C208A-MRP1 3.0 ( 0.6 (0.8) (n ) 3) 2.4 ( 0.6 (1.2) (n ) 3) C208S-MRP1 4.2 ( 0.8 (1.2) (n ) 3) 3.8 ( 0.7 (1.9) (n ) 3) C265A-MRP1 2.5 ( 0.2 (0.7) (n ) 4) 2.3 ( 0.6 (0.9) (n ) 3) C265S-MRP1 10.2 ( 0.4 (2.8)b (n ) 5) 4.0 ( 1.7 (2) (n ) 3) a The resistance of stably transfected HeLa cells was determined using a tetrazolium-based cytotoxicity assay. The relative resistance factors were obtained by dividing the IC50 values for wild-type or Cys mutant MRP1 transfected cells by the IC50 values for empty vector control transfected cells and were normalized for differences in MRP1 expression levels.
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ABCC1 p.Cys265Ser 12731862:192:807
status: NEW197 In addition, several Cys mutants did not appear fully routed to the plasma membrane, with the most severely disrupted pattern of subcellular localization being observed with the TM1 mutant Cys43Ser and the CL3 mutants Cys265Ala and Cys265Ser.
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ABCC1 p.Cys265Ser 12731862:197:232
status: NEW210 All Cys-substituted proteins were present to a certain extent in the plasma membrane of the FIGURE 7: Resistance of HeLa cells expressing Cys43Ser and Cys265Ser mutant MRP1 to sodium arsenite.
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ABCC1 p.Cys265Ser 12731862:210:151
status: NEW211 HeLa cell lines stably transfected with the pcDNA3.1(-) vector (O), pcDNA3.1(-)- MRP1K (b), and (A) pcDNA3.1(-)C43S-MRP1K (9) or (B) pcDNA3.1(-)-C265S-MRP1K (9) were exposed to sodium arsenite for 72 h at 37 °C at the concentrations indicated, and then, cell viability was measured using a tetrazolium assay. The results shown are those of a typical experiment in which each data point represents the mean (SD of quadruplicate determinations.
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ABCC1 p.Cys265Ser 12731862:211:145
status: NEW213 Data are not normalized for MRP1 expression; however, Cys265Ser-MRP1 and Cys43Ser-MRP1 were expressed at levels comparable to, and 1.5-fold higher than, WT-MRP1, respectively.
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ABCC1 p.Cys265Ser 12731862:213:54
status: NEW214 Consequently, normalization of the data would not affect the relative resistance of Cys265Ser-MRP1 and would only further emphasize the decreased arsenite resistance of Cys43Ser-MRP1.
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ABCC1 p.Cys265Ser 12731862:214:84
status: NEW237 In addition to their limited effects on organic anion transport function, the mutation of Cys residues in MSD1 and CL3 Cys had no effect on the ability of MRP1 to confer resistance to heavy metal-centered oxyanions with just two exceptions (Cys43Ser-MRP1 and Cys265Ser-MRP1).
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ABCC1 p.Cys265Ser 12731862:237:259
status: NEW253 Thus, one possible explanation for the increased arsenite resistance of Cys265Ser-MRP1 is that arsenite could be forming a complex with the mutant protein that is not held with as high affinity as WT-MRP1.
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ABCC1 p.Cys265Ser 12731862:253:72
status: NEW255 Another possible explanation is that substitution of Cys265 with Ser but not Ala results in a conformational change within CL3 that indirectly increases the efficiency with which arsenite interacts with the protein.
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ABCC1 p.Cys265Ser 12731862:255:55
status: NEW