Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4

Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.     

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.     

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd²⁺), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd²⁺ can be transported as phytochelatin‐Cd²⁺ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd²⁺ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 μM), and high (200 μM) Cd²⁺ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ?). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low‐Cd²⁺ conditions, an inorganic pyrophosphatase and a γ‐tonoplast intrinsic protein (γ‐TIP) were up‐regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance‐associated macrophage protein (NRAMP), responsible for vacuolar Fe²⁺ export was increased. CAX1a might play a role in vacuolar Cd²⁺ transport. An increase in NRAMP activity leads to a higher cytosolic Fe²⁺ concentration, which may prevent the exchange of Fe²⁺ by toxic Cd²⁺. Additionally, an ABC transporter homolog to AtMRP3 showed up‐regulation. Under high Cd²⁺ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd²⁺ conditions, was up‐regulated. Interestingly, AtMRP3 is able to partially rescue a Cd²⁺‐sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd²⁺ detoxification.     

Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation

ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.     

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1)

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of “safe-food” strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.     

Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.     

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr³²⁴ in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.     

Synthesis and biological evaluation of a series of flavone derivatives as potential radioligands for imaging the multidrug resistance-associated protein 1 (ABCC1/MRP1)

Multidrug resistance (MDR) is one of the major problems affecting the treatment of cancer. In vivo visualization and quantification of MDR proteins would be of great value to better select the therapeutic strategy. Six flavone-based compounds were synthesized and evaluated for their cytotoxic activity and MDR-reversing capacity using hMRP1 or hMDR1 overexpressing cell lines for in vitro assays. All the flavone derivatives were highly selective for hMRP1-expressing cell lines. These derivatives each used at 4muM (a non-cytotoxic concentration) enhance significantly the sensitivity of hMRP1-mediated MDR cell line toward doxorubicin toxicity. Their MDR-reversing capacity suggests that, in particular, the 4'-fluoroalkyloxy and 4'-iodo apigenin derivatives are potential new radiopharmaceuticals to visualize in vivo MRP1-mediated MDR phenomenon by PET or SPECT.     

Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT)

The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.     

S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1).

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.     

Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone.

Regulation of multidrug resistance-associated protein (MRP2) expression in response to dexamethasone (DEX) was analyzed using mainly primary rat hepatocytes. Enhanced levels of MRP2 mRNAs associated with increased amounts of a 190 kDa MRP2 were found in cultured DEX-treated hepatocytes; similarly, administration of DEX to rats (100 mg/kg, i.p.) led to a marked increase of hepatic amounts of MRP2 mRNAs. Maximal induction of MRP2 expression in DEX-treated primary hepatocytes was reached with 10(-5) M DEX, a concentration higher than that (10(-7) M) required for maximal up-regulation of tyrosine aminotransferase (TAT), a typical glucocorticoid receptor-regulated enzyme. In addition, the anti-glucocorticoid compound RU486 failed to inhibit MRP2 induction caused by DEX whereas it fully blocked that of TAT. These findings therefore demonstrate that DEX is a potent inducer of MRP2 expression in rat hepatocytes through a mechanism that seems not to involve the classical glucocorticoid receptor pathway.     

三磷酸腺苷结合盒转运体和细胞胆固醇外排

胆固醇是细胞质膜的重要组成成分。然而,过多的胆固醇累积可导致细胞中毒。异常的胆固醇胞内迁移与蓄积是造成许多心血管疾病如动脉粥样硬化的分子基础。细胞内胆固醇稳态由胆固醇的吸收、合成及外排等一系列过程调控。在哺乳动物细胞中,调节胆固醇合成、吸收和外排是维持体内胆固醇平衡的必要生理过程。本综述着重概述了三磷酸腺苷结合盒转运体(ABC)家族,如ABCA1、ABCG1、ABCG5和ABCG8的细胞功能及生理作用,以及这些转运体在调控胆固醇胞外转运中的分子机制。     

The 70-kDa peroxisomal membrane protein (PMP70) an ATP-binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of major components of peroxisomal membranes. In rodents, PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid β-oxidation enzymes. PMP70 is an ATP-binding cassette transporter, identified for the first time in intracellular membranes of eukaryotic cells. The authors' recent studies suggest that PMP70 is synthesized on free polysomes and posttranslationally inserted into peroxisomal membranes, and assembles as dimeric or oligomeric forms on peroxisomal membranes. PMP70 is suggested to be involved in metabolic transport of long-chain acyl-CoA across peroxisomal membranes.     

The structure of the multidrug resistance protein 1 (MRP1/ABCC1). crystallization and single-particle analysis

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.