Mutation of Glu521 or Glu535 in Cytoplasmic Loop 5 Causes Differential Misfolding in Multiple Domains of Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Iram, Surtaj H.; Cole, Susan P.C.

doi:10.1074/jbc.m111.310409

Cited by 28 publications

(27 citation statements)

References 43 publications

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“…In vitro experiments suggest that breast cancer related protein (BCRP) [101], CFTR (ABCC7) [102,103], MRP1 (ABCC1) [104], ABCB1 and SUR1 (ABCC8) can be rescued by small molecules.…”

Section: Introductionmentioning

confidence: 99%

Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters

Chiba

Freissmuth

Stockner

2014

Pharmacological Research

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“…In vitro experiments suggest that breast cancer related protein (BCRP) [101], CFTR (ABCC7) [102,103], MRP1 (ABCC1) [104], ABCB1 and SUR1 (ABCC8) can be rescued by small molecules.…”

Section: Introductionmentioning

confidence: 99%

Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters

Chiba

Freissmuth

Stockner

2014

Pharmacological Research

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“…Still others lead to misfolding and lower levels of MRP1 at the plasma membrane Iram and Cole, 2012). Consequently, we wondered if the discordance between the effects of the ABCC1 nsSNPs predicted in silico and our experimental data were due to limitations of the SIFT and PolyPhen algorithms and/or because the functional assays used were not sufficiently comprehensive to detect all possible phenotypic changes.…”

Section: Introductionmentioning

confidence: 99%

“…Because the functional changes detected for A989T and C1047S were the most significant among all the nsSNPs tested, we determined whether any of these changes were associated with any differences in protein conformation as reflected by a change in protease susceptibility. Thus, membrane vesicles enriched for wild-type and mutant MRP1 were digested with a range of trypsin/ protein ratios, and tryptic fragments were detected by immunoblotting with a panel of antibodies that detect epitopes in different domains of the transporter (Iram and Cole, 2012). Tryptic digests were performed in the presence and absence of S-methyl GSH because previous studies have shown that this tripeptide (and the less stable GSH) can stimulate substrate transport (and modulate inhibitor activity) as well as alter the conformation of MRP1 (Loe et al, 1998;Mao et al, 2002;Peklak-Scott et al, 2005;Ren et al, 2005;Rothnie et al, 2006;Cole, 2013).…”

Section: Bioinformatic Analyses Of Predicted Consequences Of Mrp1mentioning

confidence: 99%

“…Samples were resolved by SDS-PAGE on 4%-20% tris-glycine gradient gels (BioRad Laboratories, Hercules, CA) and immunoblotted. Full-length and tryptic fragments of MRP1 were detected using MRP1-specific mAbs MRPr1 (1:5,000), 42.4 (1:5,000), MRPm5 (1:5,000), 897.2 (1:5,000), and MRPm6 (1:5,000), whose epitopes have been mapped to amino acids 238-247, 723-732, 1063-10721318-1388, and 1511-1520, respectively (Hipfner et al, 1998Hou et al, 2000;Iram and Cole, 2012).…”

mentioning

confidence: 99%

“…Tryptic fragmentation patterns of wild-type and A989T mutant MRP1 were determined as described previously elsewhere (Rothnie et al, 2006;Iram and Cole, 2012). Briefly, membrane vesicles (2 mg of protein per lane) enriched for wild-type or A989T mutant MRP1 were incubated in hypotonic buffer (50 mM HEPES, pH 7.4) in the absence or presence of S-methylGSH (10 mM) for 30 minutes on ice.…”

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confidence: 99%

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Two Polymorphic Variants ofABCC1Selectively Alter Drug Resistance and Inhibitor Sensitivity of the Multidrug and Organic Anion Transporter Multidrug Resistance Protein 1

Conseil

Cole

2013

Drug Metab Dispos

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In this study we compared the in silico predictions of the effect of ABCC1 nonsynonymous single nucleotide polymorphisms (nsSNPs) with experimental data on MRP1 transport function and response to chemotherapeutics and multidrug resistance protein 1 (MRP1) inhibitors. Vectors encoding seven ABCC1 nsSNPs were stably expressed in human embryonic kidney (HEK) cells, and levels and localization of the mutant MRP1 proteins were determined by confocal microscopy and immunoblotting. The function of five of the mutant proteins was determined using cell-based drug and inhibitor sensitivity and efflux assays, and membrane-based organic anion transport assays. Predicted consequences of the mutations were determined by multiple bioinformatic methods. Mutants C43S and S92F were correctly routed to the HEK cell plasma membrane, but the levels were too low to permit functional characterization. In contrast, levels and membrane trafficking of R633Q, G671V, R723Q, A989T, and C1047S were similar to wild-type MRP1. In cell-based assays, all five mutants were equally effective at effluxing calcein, but only two exhibited reduced resistance to etoposide (C1047S) and vincristine (A989T; C1047S). The GSH-dependent inhibitor quinolin-5-yl)-cyclohexylmethyl]-benzamide)] was less effective at blocking calcein efflux by A989T, but in a membrane-based assay, organic anion transport by A989T and C1047S was inhibited by MRP1 modulators as well as wild-type MRP1. GSH accumulation assays suggest cellular GSH efflux by A989T and C1047S may be impaired. In conclusion, although six in silico analyses consistently predict deleterious consequences of ABCC1 nsSNPs G671V, changes in drug resistance and inhibitor sensitivity were only observed for A989T and C1047S, which may relate to GSH transport differences.

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Study on functional sites in human multidrug resistance protein 1 (hMRP1)

Han

Farooq

et al. 2021

Proteins

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Human multidrug resistance protein 1 (hMRP1) is an important member of the ATPbinding cassette (ABC) transporter superfamily. It can extrude a variety of anticancer drugs and physiological organic anions across the plasma membrane, which is activated by substrate binding, and is accompanied by large-scale cooperative movements between different domains. Currently, it remains unclear completely about how the specific interactions between hMRP1 and its substrate are and which critical residues are responsible for allosteric signal transduction. To the end, we first construct an inward-facing state of hMRP1 using homology modeling method, and then dock substrate proinflammatory agent leukotriene C4 (LTC4) to hMRP1 pocket. The result manifests LTC4 interacts with two parts of hMRP1 pocket, namely the positively charged pocket (P pocket) and hydrophobic pocket (H pocket), similar to its binding mode with bMRP1 (bovine MRP1). Additionally, we use the Gaussian network model (GNM)-based thermodynamic method proposed by us to identify the key residues whose perturbations markedly alter their binding free energy. Here the conventional GNM is improved with covalent/non-covalent interactions and secondary structure information considered (denoted as sscGNM). In the result, sscGNM improves the flexibility prediction, especially for the nucleotide binding domains with rich kinds of secondary structures. The 46 key residue clusters located in different subdomains are identified which are highly consistent with experimental observations. Furtherly, we explore the long-range cooperation within the transporter. This study is helpful for strengthening the understanding of the work mechanism in ABC exporters and can provide important information to scientists in drug design studies.binding mode, Gaussian network model, hMRP1, key residues, thermodynamic cycle | INTRODUCTIONMultidrug resistance (MDR) to chemotherapy is a major obstacle in the treatment of cancer patients. Human multidrug resistance protein 1 (hMRP1), a member of the ATP-binding cassette (ABC) transporter superfamily, can export a wide variety of anticancer drugs such as vincristine, etoposide, anthracyclines, methotrexate, and physiological organic anions antioxidants GSH and proinflammatory agent leukotriene C4 (LTC4) across the cell membrane utilizing the energy from ATP binding and hydrolysis. 1 Studies have shown that the process of substrate transport is accompanied by large-scale cooperative motions between spatially separated subdomains. 2 Thus, there must exist a network of key residues mediating long-range signal transmission and allosteric coupling. 2,3 Identification of key functional residues is very important not only for an "in-depth" understanding of the mechanism of signal transmission but also for the structure-based drug design.

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Mutation of Glu521 or Glu535 in Cytoplasmic Loop 5 Causes Differential Misfolding in Multiple Domains of Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Cited by 28 publications

References 43 publications

Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters

Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters

Two Polymorphic Variants ofABCC1Selectively Alter Drug Resistance and Inhibitor Sensitivity of the Multidrug and Organic Anion Transporter Multidrug Resistance Protein 1

Study on functional sites in human multidrug resistance protein 1 (hMRP1)

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