ABCMdb

ABCC1 p.Cys148Ser

Predicted by SNAP2:	A: N (72%), D: D (75%), E: D (75%), F: N (53%), G: D (59%), H: D (75%), I: D (71%), K: D (80%), L: D (53%), M: D (75%), N: D (66%), P: D (80%), Q: D (66%), R: D (80%), S: N (57%), T: N (72%), V: D (63%), W: D (85%), Y: D (71%),
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] 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). Login to comment
130 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. Login to comment
132 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. Login to comment
133 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. Login to comment
156 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. Login to comment
185 The lowest expression levels were observed with the MSD1 mutants Cys85Ser, Cys148Ser, and Cys190Ser, which were FIGURE 6: Uptake of [3H]LTC4, [3H]E217 G, and [3H]GSH by inside-out membrane vesicles prepared from wild-type and Cys-substituted MRP1 transfected HeLa cells. Login to comment

[hide] Transverse and tangential orientation of predicted... Eur Biophys J. 2011 Sep;40(9):1043-60. Epub 2011 Jun 24. de Foresta B, Vincent M, Garrigos M, Gallay J
Transverse and tangential orientation of predicted transmembrane fragments 4 and 10 from the human multidrug resistance protein (hMRP1/ABCC1) in membrane mimics.
Eur Biophys J. 2011 Sep;40(9):1043-60. Epub 2011 Jun 24., [PMID:21701864]

Abstract [show]
The human multidrug-resistance-associated protein 1 (hMRP1/ABCC1) belongs to the large ATP-binding cassette transporter superfamily. In normal tissues, hMRP1 is involved in tissue defense, whereas, in cancer cells, it is overproduced and contributes to resistance to chemotherapy. We previously investigated the folding properties of the predicted transmembrane fragments (TM) TM16, and TM17 from membrane-spanning domain 2 (MSD2). These TMs folded only partially as an alpha-helix and were located in the polar headgroup region of detergent micelles used as membrane mimics (Vincent et al. in Biochim Biophys Acta 1768:538-552, 2007; de Foresta et al. in Biochim Biophys Acta 1798:401-414, 2010). We have now extended these studies to TM4 and TM10, from MSD0 and MSD1, respectively. TM10 may be involved in the substrate translocation pathway whereas the role of TM4 is less predictable, because few studies have focused on MSD0, a domain present in some hMRP1 homologs only. Each TM contained a single Trp residue (W142 or W553) acting as an intrinsic fluorescent probe. The location and dynamics of the TMs in dodecylphosphocholine (DPC) or n-dodecyl-beta-D: -maltoside (DDM) micelles were studied by Trp steady-state and time-resolved fluorescence, including quenching experiments. Overall TM structure was analyzed by far-UV circular dichroism studies in detergent micelles and TFE. TM10 behaved similarly to TM16 and TM17, with an interfacial location in micelles consistent with a possible role in lining the transport pore. By contrast, TM4 behaved like a classical TM fragment with a high alpha-helical content, and its transmembrane insertion did not require its interaction with other TMs.

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No. Sentence Comment
79 Mutations introduced: C148S in mTM4, C555S and C563S in mTM10 Materials and methods Chemicals We obtained n-dodecyl-b-D-maltoside (DDM) from Calbiochem. Login to comment