Science.gov

Sample records for abc transporter p-glycoprotein

  1. Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling.

    PubMed

    Jones, P M; George, A M

    2005-04-30

    Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein. PMID:15826647

  2. Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier

    PubMed Central

    Miller, David S.

    2010-01-01

    ATP-binding cassette (ABC) transporters are important, selective elements of the blood-brain barrier. They line the luminal plasma membrane of the brain capillary endothelium, facing the vascular space, both protecting the CNS from entry of neurotoxicants and limiting access of therapeutic drugs to the brain parenchyma. Recent studies highlight the multiple signaling pathways through which the expression and activity of P-glycoprotein and other ABC transporters are modulated in response to xenobiotics, stress and disease. They show that increased transporter expression occurs in response to signals that activate specific transcription factors including, PXR, CAR, NF-κB and AP-1, and reduced transporter activity occurs rapidly and reversibly in response to signaling through Src kinase, protein kinase C and estrogen receptors. A detailed understanding of such regulation can provide the basis for improved neuroprotection and enhanced therapeutic drug delivery to the brain. PMID:20417575

  3. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that

  4. Haemonchus contortus P-Glycoproteins Interact with Host Eosinophil Granules: A Novel Insight into the Role of ABC Transporters in Host-Parasite Interaction

    PubMed Central

    Issouf, Mohamed; Guégnard, Fabrice; Koch, Christine; Le Vern, Yves; Blanchard-Letort, Alexandra; Che, Hua; Beech, Robin N.; Kerboeuf, Dominique; Neveu, Cedric

    2014-01-01

    Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response. PMID:24498376

  5. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  6. P-Glycoprotein Transport of Neurotoxic Pesticides.

    PubMed

    Lacher, Sarah E; Skagen, Kasse; Veit, Joachim; Dalton, Rachel; Woodahl, Erica L

    2015-10-01

    P-glycoprotein (P-gp) has been associated with a number of neurodegenerative diseases, including Parkinson's disease, although the mechanisms remain unclear. Altered transport of neurotoxic pesticides has been proposed in Parkinson's disease, but it is unknown whether these pesticides are P-gp substrates. We used three in vitro transport models, stimulation of ATPase activity, xenobiotic-induced cytotoxicity, and inhibition of rhodamine-123 efflux, to evaluate P-gp transport of diazinon, dieldrin, endosulfan, ivermectin, maneb, 1-methyl-4-phenyl-4-phenylpyridinium ion (MPP(+)), and rotenone. Diazinon and rotenone stimulated ATPase activity in P-gp-expressing membranes, with Vmax values of 22.4 ± 2.1 and 16.8 ± 1.0 nmol inorganic phosphate/min per mg protein, respectively, and Km values of 9.72 ± 3.91 and 1.62 ± 0.51 µM, respectively, compared with the P-gp substrate verapamil, with a Vmax of 20.8 ± 0.7 nmol inorganic phosphate/min per mg protein and Km of 0.871 ± 0.172 μM. None of the other pesticides stimulated ATPase activity. We observed an increased resistance to MPP(+) and rotenone in LLC-MDR1-WT cells compared with LLC-vector cells, with 15.4- and 2.2-fold increases in EC50 values, respectively. The resistance was reversed in the presence of the P-gp inhibitor verapamil. None of the other pesticides displayed differential cytotoxicity. Ivermectin was the only pesticide to inhibit P-gp transport of rhodamine-123, with an IC50 of 0.249 ± 0.048 μM. Our data demonstrate that dieldrin, endosulfan, and maneb are not P-gp substrates or inhibitors. We identified diazinon, MPP(+), and rotenone as P-gp substrates, although further investigation is needed to understand the role of P-gp transport in their disposition in vivo and associations with Parkinson's disease.

  7. Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

    PubMed

    Clay, Adam T; Lu, Peihua; Sharom, Frances J

    2015-11-01

    The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

  8. Identification of ABC transporters in Sarcoptes scabiei.

    PubMed

    Mounsey, K E; Holt, D C; McCarthy, J; Walton, S F

    2006-06-01

    We have identified and partially sequenced 8 ABC transporters from an EST dataset of Sarcoptes scabiei var. hominis, the causative agent of scabies. Analysis confirmed that most of the known ABC subfamilies are represented in the EST dataset including several members of the multidrug resistance protein subfamily (ABC-C). Although P-glycoprotein (ABC-B) sequences were not found in the EST dataset, a partial P-glycoprotein sequence was subsequently obtained using a degenerate PCR strategy and library screening. Thus a total of 9 potential S. scabiei ABC transporters representing the subfamilies A, B, C, E, F and H have been identified. Ivermectin is currently used in the treatment of hyper-infested (crusted) scabies, and has also been identified as a potentially effective acaricide for mass treatment programmes in scabies-endemic communities. The observation of clinical and in vitro ivermectin resistance in 2 crusted scabies patients who received multiple treatments has raised serious concerns regarding the sustainability of such programmes. One possible mechanism for ivermectin resistance is through ABC transporters such as P-glycoprotein. This work forms an important foundation for further studies to elucidate the potential role of ABC transporters in ivermectin resistance of S. scabiei.

  9. Multiple Drug Transport Pathways through Human P-Glycoprotein.

    PubMed

    McCormick, James W; Vogel, Pia D; Wise, John G

    2015-07-21

    P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

  10. Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport.

    PubMed

    Thalhammer, T; Stapf, V; Gajdzik, L; Graf, J

    1994-04-01

    This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

  11. A single active catalytic site is sufficient to promote transport in P-glycoprotein.

    PubMed

    Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, Imre J; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

    2016-04-27

    P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

  12. A single active catalytic site is sufficient to promote transport in P-glycoprotein

    PubMed Central

    Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, Imre J.; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

    2016-01-01

    P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis. PMID:27117502

  13. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice.

    PubMed

    Wang, Xueqian; Cabrera, Robert M; Li, Yue; Miller, David S; Finnell, Richard H

    2013-03-01

    Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.

  14. Haemonchus contortus P-glycoprotein-2: in situ localisation and characterisation of macrocyclic lactone transport.

    PubMed

    Godoy, Pablo; Lian, Jing; Beech, Robin N; Prichard, Roger K

    2015-01-01

    Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm's intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus.

  15. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    SciTech Connect

    Piwnica-Worms, D.; Vallabhaneni, V.R.; Kronauge, J.F.

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.

  16. P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

    PubMed

    Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

    2010-03-01

    We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. PMID:20004641

  17. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice

    PubMed Central

    Wang, Xueqian; Cabrera, Robert M.; Li, Yue; Miller, David S.; Finnell, Richard H.

    2013-01-01

    Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.—Wang, X., Cabrera, R. M., Li, Y., Miller, D. S., Finnell, R. H. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. PMID:23212123

  18. Inhibition of rhodamine 123 secretion by cyclosporin A as a model of P-glycoprotein mediated transport in liver.

    PubMed

    Stapf, V; Thalhammer, T; Huber-Huber, R; Felberbauer, F; Gajdzik, L; Graf, J

    1994-01-01

    The interaction between P-glycoprotein modulators and P-glycoprotein mediated transport was investigated using rhodamine 123 in the isolated perfused rat liver of a mutant (TR-) rat strain. TR- rats, deficient in the canalicular multispecific anion transport system, are unable to extrude organic anions (glucuronides) and therefore excrete solely unconjugated rhodamine 123 via P-glycoprotein. Cyclosporin A, a modulator of multidrug resistance in tumor cells, inhibited the biliary secretion of rhodamine 123 dose dependently in a non-competitive manner. Both cyclosporin A and rhodamine inhibited photoaffinity labeling of immunoprecipitated P-glycoprotein with azidopine, indicating binding to hepatic P-glycoprotein. Our results indicate that monitoring the biliary rhodamine 123 secretion in the isolated perfused liver of TR- rats offers a new system for testing modulators of P-glycoprotein like cyclosporin A.

  19. Thermodynamics of ABC transporters.

    PubMed

    Zhang, Xuejun C; Han, Lei; Zhao, Yan

    2016-01-01

    ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion. We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.

  20. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  1. Cloning of two novel ABC transporters mapping on human chromosome 9

    SciTech Connect

    Luciani, M.F.; Savary, S.; Chimini, G. ); Denizot, F. ); Mattei, M.G. )

    1994-05-01

    The family of ATP binding cassette (ABC) transporters or traffic ATPases is composed of several membrane-associated proteins that transport a great variety of solutes across cellular membranes. Two novel mammalian members of the family, ABC1 and ABC2, have been identified by a PCR-based approach. They belong to a group of traffic ATPases encoded as a single multifunctional protein, such as CFTR, STE 6, and P-glycoproteins. Their peculiar structural features and close relationship to ABC transporters involved in nodulation suggest that ABC1 and ABC2 define a novel subgroup of mammalian traffic ATPases. 51 refs., 7 figs.

  2. ABC transporters in fish species: a review

    PubMed Central

    Ferreira, Marta; Costa, Joana; Reis-Henriques, Maria A.

    2014-01-01

    ATP-binding cassette (ABC) proteins were first recognized for their role in multidrug resistance (MDR) in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR). In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is necessary to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps), multidrug-resistance-associated proteins (MRPs 1-5) and breast cancer resistance associated protein (BCRP). In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of the detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants that can act as chemosensitizers, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in (1) regulation and functioning of ABC proteins; (2) cooperation with phase I and II biotransformation enzymes; and (3) ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clearly suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish to underlay the mechanism to consider their use as

  3. Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    PubMed Central

    Souza, Paloma S.; Madigan, James P.; Gillet, Jean-Pierre; Kapoor, Khyati; Ambudkar, Suresh V.; Maia, Raquel C.; Gottesman, Michael M.; Fung, King Leung

    2015-01-01

    Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells. PMID:26101157

  4. Structural diversity of ABC transporters

    PubMed Central

    ter Beek, Josy; Guskov, Albert

    2014-01-01

    ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity. PMID:24638992

  5. Edoxaban transport via P-glycoprotein is a key factor for the drug's disposition.

    PubMed

    Mikkaichi, Tsuyoshi; Yoshigae, Yasushi; Masumoto, Hiroshi; Imaoka, Tomoki; Rozehnal, Veronika; Fischer, Thomas; Okudaira, Noriko; Izumi, Takashi

    2014-04-01

    Edoxaban (the free base of DU-176b), an oral direct factor Xa inhibitor, is mainly excreted unchanged into urine and feces. Because active membrane transport processes such as active renal secretion, biliary excretion, and/or intestinal secretion, and the incomplete absorption of edoxaban after oral administration have been observed, the involvement of drug transporters in the disposition of edoxaban was investigated. Using a bidirectional transport assay in human colon adenocarcinoma Caco-2 cell monolayers, we observed the vectorial transport of [(14)C]edoxaban, which was completely inhibited by verapamil, a strong P-glycoprotein (P-gp) inhibitor. In an in vivo study, an increased distribution of edoxaban to the brain was observed in Mdr1a/1b knockout mice when compared with wild-type mice, indicating that edoxaban is a substrate for P-gp. However, there have been no observations of significant transport of edoxaban by renal or hepatic uptake transporters, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, or organic anion transporting polypeptide (OATP)1B1. Edoxaban exhibited no remarkable inhibition of OAT1, OAT3, OCT1, OCT2, OATP1B1, OATP1B3, or P-gp up to 30 μM; therefore, the risk of clinical drug-drug interactions due to any edoxaban-related transporter inhibition seems to be negligible. Our results demonstrate that edoxaban is a substrate of P-gp but not of other major uptake transporters tested. Because metabolism is a minor contributor to the total clearance of edoxaban and strong P-gp inhibitors clearly impact edoxaban transport, the P-gp transport system is a key factor for edoxaban's disposition.

  6. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  7. Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies.

    PubMed

    Sharom, F J; Liu, R; Romsicki, Y; Lu, P

    1999-12-01

    The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy.

  8. Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Ernst, S.; Verhalen, B.; Zarrabi, N.; Wilkens, S.; Börsch, M.

    2011-03-01

    In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

  9. The human P-glycoprotein transporter enhances the type I interferon response to Listeria monocytogenes infection.

    PubMed

    Sigal, Nadejda; Kaplan Zeevi, Millie; Weinstein, Shiri; Peer, Dan; Herskovits, Anat A

    2015-06-01

    Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen Listeria monocytogenes. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against L. monocytogenes bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by L. monocytogenes during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted L. monocytogenes or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps.

  10. Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

    PubMed Central

    Chufan, Eduardo E.; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T.; Durell, Stewart R.; Ambudkar, Suresh V.

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate

  11. Blonanserin, a novel atypical antipsychotic agent not actively transported as substrate by P-glycoprotein.

    PubMed

    Inoue, Tomoko; Osada, Kenichi; Tagawa, Masaaki; Ogawa, Yuriko; Haga, Toshiaki; Sogame, Yoshihisa; Hashizume, Takanori; Watanabe, Takashi; Taguchi, Atsushi; Katsumata, Takashi; Yabuki, Masashi; Yamaguchi, Noboru

    2012-10-01

    Although blonanserin, a novel atypical antipsychotic agent with dopamine D(2)/serotonin 5-HT(2A) antagonistic properties, displays good brain distribution, the mechanism of this distribution has not been clarified. P-glycoprotein [(P-gp) or multidrug resistance protein 1 (MDR1)] is an efflux transporter expressed in the brain and plays an important role in limiting drug entry into the central nervous system (CNS). In particular, P-gp can affect the pharmacokinetics and efficacy of antipsychotics, and exacerbate or soothe their adverse effects. In this study, we conducted in vitro and in vivo experiments to determine whether blonanserin is a P-gp substrate. Risperidone and its active metabolite 9-hydroxyrisperidone, both of which are P-gp substrates, were used as reference drugs. Affinity of blonanserin, risperidone, and 9-hydroxyrisperidone for P-gp was evaluated by in vitro transcellular transport across LLC-PK1, human MDR1 cDNA-transfected LLC-PK1 (LLC-MDR1), and mouse Mdr1a cDNA-transfected LLC-PK1 (LLC-Mdr1a). In addition, pharmacokinetic parameters in the brain and plasma (B/P ratio) of test compounds were measured in mdr1a/1b knockout (KO) and wild-type (WT) mice. The results of in vitro experiments revealed that P-gp does not actively transport blonanserin as a substrate in humans or mice. In addition, blonanserin displayed comparable B/P ratios in KO and WT mice, whereas B/P ratios of risperidone and 9-hydroxyrisperidone differed markedly in these animals. Our results indicate that blonanserin is not a P-gp substrate and therefore its brain distribution is unlikely to be affected by this transporter.

  12. Characterizing the binding interactions between P-glycoprotein and eight known cardiovascular transport substrates

    PubMed Central

    Jagodinsky, Justin C; Akgun, Ugur

    2015-01-01

    The multidrug efflux pump P-glycoprotein (Pgp) is upregulated in cardiomyocytes following chronic ischemia from infarction and hypoxia caused by sleep apnea. This report summarizes the molecular dynamic studies performed on eight cardiovascular drugs to determine their corresponding binding sites on mouse Pgp. Selected Pgp transport ligands include: Amiodarone, Bepridil, Diltiazem, Dipyridamole, Nicardipine, Nifedipine, Propranolol, and Quinidine. Extensive molecular dynamic equilibration simulations were performed to determine drug docking interactions. Distinct binding sites were not observed, but rather a binding belt was seen with multiple residues playing a role in each studied drug's stable docking. Three key drug–protein interactions were identified: hydrogen bonding, hydrophobic packing, and the formation of a “cage” of aromatic residues around the drug. After drug stabilization, water molecules were observed to leak into the binding belt and condense around the drug. Water influx into the binding domain of Pgp may play a role in catalytic transition and drug expulsion. The cytoplasmic recruitment theory was also tested, and the drugs were observed to interact with conserved loops of residues with a strong affinity. A free energy change of astronomical value is required to recruit the drug from the cytoplasm to the binding belt within the transmembrane domain of Pgp. PMID:25729581

  13. In vitro and in vivo P-glycoprotein transport characteristics of rivaroxaban.

    PubMed

    Gnoth, Mark Jean; Buetehorn, Ulf; Muenster, Uwe; Schwarz, Thomas; Sandmann, Steffen

    2011-07-01

    Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated. Furthermore, the inhibitory effect of rivaroxaban toward P-gp was determined. Rivaroxaban exhibited high permeability and polarized transport across Caco-2 cells. Rivaroxaban was shown to be a substrate for, but not an inhibitor of, P-gp. Of a set of potential P-gp inhibitors, ketoconazole and ritonavir, but not clarithromycin or erythromycin, inhibited P-gp-mediated transport of rivaroxaban, with half-maximal inhibitory concentration values in the range of therapeutic plasma concentrations. These findings are in line with observed area under the plasma concentration-time curve increases in clinical drug-drug interaction studies indicating a possible involvement of P-gp in the distribution and excretion of rivaroxaban. In vivo studies in wild-type and P-gp double-knockout mice demonstrated that the impact of P-gp alone on the pharmacokinetics of rivaroxaban is minor. However, in P-gp double-knockout mice, a slight increase in brain concentrations and decreased excretion into the gastrointestinal tract were observed compared with wild-type mice. These studies also demonstrated that brain penetration of rivaroxaban is fairly low. In addition to P-gp, a further transport protein might be involved in the secretion of rivaroxaban.

  14. P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

    PubMed

    Ballent, M; Wilkens, M R; Maté, L; Muscher, A S; Virkel, G; Sallovitz, J; Schröder, B; Lanusse, C; Lifschitz, A

    2013-12-01

    The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

  15. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

    PubMed

    Evans, David C; O'Connor, Desmond; Lake, Brian G; Evers, Raymond; Allen, Christopher; Hargreaves, Richard

    2003-07-01

    "Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity. PMID:12814962

  16. ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal

    PubMed Central

    Choi, Cheol-Hee

    2005-01-01

    One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC) transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein. PMID:16202168

  17. Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli fructus.

    PubMed

    Yoshida, Naoko; Takagi, Akiyoshi; Kitazawa, Hidenori; Kawakami, Junichi; Adachi, Isao

    2005-12-01

    Citrus (rutaceous) herbs are often used in traditional medicine and Japanese cuisine and can be taken concomitantly with conventional medicine. In this study, the effect of various citrus-herb extracts on P-glycoprotein (P-gp)-mediated transport was examined in vitro to investigate a possible interaction with P-gp substrates. Component monoterpenoids of the essential oil in Zanthoxyli fructus was screened to find novel P-gp inhibitors. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used. Cellular accumulation of [3H]digoxin was measured in the presence or absence of P-gp inhibitors or test samples. Aurantii fructus, Evodiae fructus, Aurantii fructus immaturus, Aurantii nobilis pericarpium, Phellodendri cortex, and Zanthoxyli fructus were extracted with hot water (decocted) and then fractionated with ethyl acetate. The cell to medium ratio of [3H]digoxin accumulation increased significantly in the presence of the decoction of Evodiae fructus, Aurantii nobilis pericarpium, and Zanthoxyli fructus, and the ethyl acetate fraction of all citrus herbs used. The ethyl acetate fraction of Zanthoxyli fructus exhibited the strongest inhibition of P-gp among tested samples with an IC50 value of 166 microg/mL. Then its component monoterpenoids, geraniol, geranyl acetate, (R)-(+)-limonene, (R)-(+)-linalool, citronellal, (R)-(+)-citronellal, DL-citronellol, (S)-(-)-beta-citronellol, and cineole, were screened. (R)-(+)-citronellal and (S)-(-)-beta-citronellol inhibited P-gp with IC50 values of 167 microM and 504 microM, respectively. These findings suggest that Zanthoxyli fructus may interact with P-gp substrates and that some monoterpenoids with the relatively lower molecular weight of about 150 such as (R)-(+)-citronellal can be potent inhibitors of P-gp.

  18. Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli Fructus

    SciTech Connect

    Yoshida, Naoko; Takagi, Akiyoshi; Kitazawa, Hidenori; Kawakami, Junichi . E-mail: kawakami-tym@umin.ac.jp; Adachi, Isao

    2005-12-01

    Citrus (rutaceous) herbs are often used in traditional medicine and Japanese cuisine and can be taken concomitantly with conventional medicine. In this study, the effect of various citrus-herb extracts on P-glycoprotein (P-gp)-mediated transport was examined in vitro to investigate a possible interaction with P-gp substrates. Component monoterpenoids of the essential oil in Zanthoxyli Fructus was screened to find novel P-gp inhibitors. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used. Cellular accumulation of [{sup 3}H]digoxin was measured in the presence or absence of P-gp inhibitors or test samples. Aurantii Fructus, Evodiae Fructus, Aurantii Fructus Immaturus, Aurantii Nobilis Pericarpium, Phellodendri Cortex, and Zanthoxyli Fructus were extracted with hot water (decocted) and then fractionated with ethyl acetate. The cell to medium ratio of [{sup 3}H]digoxin accumulation increased significantly in the presence of the decoction of Evodiae Fructus, Aurantii Nobilis Pericarpium, and Zanthoxyli Fructus, and the ethyl acetate fraction of all citrus herbs used. The ethyl acetate fraction of Zanthoxyli Fructus exhibited the strongest inhibition of P-gp among tested samples with an IC{sub 5} value of 166 {mu}g/mL. Then its component monoterpenoids, geraniol, geranyl acetate (R)-(+)-limonene, (R)-(+)-linalool, citronellal (R)-(+)-citronellal, DL-citronellol (S)-(-)-{beta}-citronellol, and cineole, were screened. (R)-(+)-citronellal and (S)-(-)-{beta}-citronellol inhibited P-gp with IC{sub 5} values of 167 {mu}M and 504 {mu}M, respectively. These findings suggest that Zanthoxyli Fructus may interact with P-gp substrates and that some monoterpenoids with the relatively lower molecular weight of about 150 such as (R)-(+)-citronellal can be potent inhibitors of P-gp.

  19. The role of ABC transporters in drug resistance, metabolism and toxicity.

    PubMed

    Glavinas, Hristos; Krajcsi, Péter; Cserepes, Judit; Sarkadi, Balázs

    2004-01-01

    ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.

  20. P-glycoproteins and other multidrug resistance transporters in the pharmacology of anthelmintics: Prospects for reversing transport-dependent anthelmintic resistance

    PubMed Central

    Lespine, Anne; Ménez, Cécile; Bourguinat, Catherine; Prichard, Roger K.

    2011-01-01

    Parasitic helminths cause significant disease in animals and humans. In the absence of alternative treatments, anthelmintics remain the principal agents for their control. Resistance extends to the most important class of anthelmintics, the macrocyclic lactone endectocides (MLs), such as ivermectin, and presents serious problems for the livestock industries and threatens to severely limit current parasite control strategies in humans. Understanding drug resistance is important for optimizing and monitoring control, and reducing further selection for resistance. Multidrug resistance (MDR) ABC transporters have been implicated in ML resistance and contribute to resistance to a number of other anthelmintics. MDR transporters, such as P-glycoproteins, are essential for many cellular processes that require the transport of substrates across cell membranes. Being overexpressed in response to chemotherapy in tumour cells and to ML-based treatment in nematodes, they lead to therapy failure by decreasing drug concentration at the target. Several anthelmintics are inhibitors of these efflux pumps and appropriate combinations can result in higher treatment efficacy against parasites and reversal of resistance. However, this needs to be balanced against possible increased toxicity to the host, or the components of the combination selecting on the same genes involved in the resistance. Increased efficacy could result from modifying anthelmintic pharmacokinetics in the host or by blocking parasite transporters involved in resistance. Combination of anthelmintics can be beneficial for delaying selection for resistance. However, it should be based on knowledge of resistance mechanisms and not simply on mode of action classes, and is best started before resistance has been selected to any member of the combination. Increasing knowledge of the MDR transporters involved in anthelmintic resistance in helminths will play an important role in allowing for the identification of markers

  1. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier

    PubMed Central

    Li, Yangfang; Wu, Qian; Li, Chen; Liu, Ling; Du, Kun; Shen, Jin; Wu, Yuqin; Zhao, Xiaofen; Zhao, Mei; Bao, Lingyun; Gao, Jin; Keep, Richard F.; Xiang, Jianming

    2016-01-01

    While the blood-brain barrier (BBB) protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC) transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP) but not those overexpressing human P-gp (MDCKII-MDR cells) had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated. PMID:27300692

  2. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    PubMed Central

    Lopez, Dioxelis; Martinez-Luis, Sergio

    2014-01-01

    P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters. PMID:24451193

  3. Identification of Possible Binding Sites for Morphine and Nicardipine on the Multidrug Transporter P-Glycoprotein Using Umbrella Sampling Techniques.

    PubMed

    Subramanian, Nandhitha; Condic-Jurkic, Karmen; Mark, Alan E; O'Mara, Megan L

    2015-06-22

    The multidrug transporter P-glycoprotein (P-gp) is central to the development of multidrug resistance in cancer. While residues essential for transport and binding have been identified, the location, composition, and specificity of potential drug binding sites are uncertain. Here molecular dynamics simulations are used to calculate the free energy profile for the binding of morphine and nicardipine to P-gp. We show that morphine and nicardipine primarily interact with key residues implicated in binding and transport from mutational studies, binding at different but overlapping sites within the transmembrane pore. Their permeation pathways were distinct but involved overlapping sets of residues. The results indicate that the binding location and permeation pathways of morphine and nicardipine are not well separated and cannot be considered as unique. This has important implications for our understanding of substrate uptake and transport by P-gp. Our results are independent of the choice of starting structure and consistent with a range of experimental studies.

  4. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

  5. P-Glycoprotein-Mediated Transport of Itraconazole across the Blood-Brain Barrier

    PubMed Central

    Miyama, Tetsuo; Takanaga, Hitomi; Matsuo, Hirotami; Yamano, Katsuhiro; Yamamoto, Koujirou; Iga, Tatsuji; Naito, Mikihiko; Tsuruo, Takashi; Ishizuka, Hitoshi; Kawahara, Yukinori; Sawada, Yasufumi

    1998-01-01

    The mechanism for the accumulation of itraconazole (ITZ) in its elimination from the brain was studied in rats and mice. The concentration of ITZ in liver tissue declined in parallel with the plasma ITZ concentration until 24 h after intravenous injection of the drug (half-life, 5 h); however, the ITZ in brain tissue rapidly disappeared (half-life, 0.4 h). The time profiles of the brain/plasma ITZ concentration ratio (Kp value) showed a marked overshooting, and the Kp value increased with increasing dose; these phenomena were not observed in the liver tissue. This finding indicates the occurrence of a nonlinear efflux of ITZ from the brain to the blood. Moreover, based on a pharmacokinetic model which hypothesized processes for both nonlinear and linear effluxes of ITZ from the brain to the blood, we found that the efflux rate constant in the saturable process was approximately sevenfold larger than that in the nonsaturable process. The Kp value for the brain tissue was significantly increased in the presence of ketoconazole or verapamil. The brain Kp value for mdr1a knockout mice was also significantly increased compared with that of control mice. Moreover, the uptake of vincristine or vinblastine, both of which are substrates of the P glycoprotein (P-gp), into mouse brain capillary endothelial cells was also significantly increased by ITZ or verapamil. In conclusion, P-gp in the brain capillary endothelial cells participates in a process of active efflux of ITZ from the brain to the blood at the blood-brain barrier, and ITZ can be an inhibitor of various substrates of P-gp. PMID:9661014

  6. Mapping the functional yeast ABC transporter interactome

    PubMed Central

    Snider, Jamie; Hanif, Asad; Lee, Mid Eum; Jin, Ke; Yu, Analyn R.; Graham, Chris; Chuk, Matthew; Damjanovic, Dunja; Wierzbicka, Marta; Tang, Priscilla; Balderes, Dina; Wong, Victoria; Jessulat, Matthew; Darowski, Katelyn D.; Luis, Bryan-Joseph San; Shevelev, Igor; Sturley, Stephen L; Boone, Charles; Greenblatt, Jack F.; Zhang, Zhaolei; Paumi, Christian M.; Babu, Mohan; Park, Hay-Oak; Michaelis, Susan; Stagljar, Igor

    2013-01-01

    ABC transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used Membrane Yeast Two-Hybrid (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated interactome. We show that ABC transporters physically associate with proteins involved in a surprisingly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters. PMID:23831759

  7. Bioinformatic survey of ABC transporters in dermatophytes.

    PubMed

    Gadzalski, Marek; Ciesielska, Anita; Stączek, Paweł

    2016-01-15

    ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity. PMID:26524502

  8. Caco-2 cell methodology and inhibition of the P-glycoprotein transport of digoxin by Aloe vera juice.

    PubMed

    Djuv, Ane; Nilsen, Odd Georg

    2008-12-01

    The aims of this study were to carry out a thorough quality control setup for essential Caco-2 cell characteristics in P-glycoprotein (P-gp) inhibition studies and to explore if Aloe vera juice (AVJ) inhibits the bidirectional transport of the P-gp substrate digoxin (30 nm). Seven AVJ concentrations (0.00001-1.0 mg/mL), anticipated to cover a clinically relevant range, were tested and digoxin apparent permeability coefficients (Papp), net Papp values (Papp(Net)) and net flux values (J(Net)) were calculated. Relevant validation parameters for P-gp inhibition studies in Caco-2 cells are suggested to include, as a minimum, an assay linearity test with and without a known P-gp inhibitor, cell cytotoxicity testing (MTT-test) for substrates and inhibitors, and cell integrity testing by TEER and mannitol transport measurements. The question is also raised whether a minimum effect of a reference P-gp inhibitor as verapamil should be demanded. Cell cytotoxicity was seen for digoxin at concentrations >or=3 microM and for AVJ at 10 mg/mL. AVJ did not inhibit the P-gp transport of digoxin in any of the concentrations tested. This indicates that AVJ is no inhibitor of the P-gp mediated transport of digoxin in vitro if AVJ is present in clinically relevant concentrations.

  9. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2016-01-01

    The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology. PMID:27043281

  10. Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models

    PubMed Central

    Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy; Wilkens, Stephan; Börsch, Michael

    2013-01-01

    Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present. PMID:23891547

  11. Opioids and efflux transporters. Part 4: influence of N-substitution on P-glycoprotein substrate activity of noroxymorphone analogues.

    PubMed

    Metcalf, Matthew D; Rosicky, Andrew D; Hassan, Hazem E; Eddington, Natalie D; Coop, Andrew; Cunningham, Christopher W; Mercer, Susan L

    2014-08-01

    The efflux transporter protein P-glycoprotein (P-gp) is capable of affecting the central distribution of diverse neurotherapeutics, including opioid analgesics, through their active removal from the brain. P-gp located at the blood brain barrier has been implicated in the development of tolerance to opioids and demonstrated to be up-regulated in rats tolerant to morphine and oxycodone. We have previously examined the influence of hydrogen-bonding oxo-substitutents on the P-gp-mediated efflux of 4,5-epoxymorphinan analgesics, as well as that of N-substituted analogues of meperidine. Structure-activity relationships (SAR) governing N-substituent effects on opioid efficacy is well-established, however the influence of such structural modifications on P-gp-mediated efflux is unknown. Here, we present SAR describing P-gp recognition of a short series of N-modified 4,5-epoxymorphinans. Oxymorphone, naloxone, naltrexone, and nalmexone all failed to demonstrate P-gp substrate activity, indicating these opioid scaffolds contain structural features that preclude recognition by the transporter. These results are examined using mathematical molecular modeling and discussed in comparison to other opioid scaffolds bearing similar N-substituents. PMID:24915880

  12. Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

    2016-02-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. PMID:26686578

  13. Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

    2016-02-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

  14. Disrupting P-glycoprotein function in clinical settings: what can we learn from the fundamental aspects of this transporter?

    PubMed Central

    Chung, Francisco S; Santiago, Jayson S; Jesus, Miguel Francisco M De; Trinidad, Camille V; See, Melvin Floyd E

    2016-01-01

    P-glycoprotein is one of the most well-studied drug transporters, significant for its role in cancer multiple drug resistance. However, using P-gp inhibitors with the aim of enhancing the therapeutic efficacy of anti-cancer drugs has led to disappointing outcomes. Furthermore, several lead compounds suggested by in vitro and pre-clinical studies have shown variable pharmacokinetics and therapeutic efficacies when applied in the clinical setting. This review will highlight the need to revisit a sound approach to better design and apply P-gp inhibitors in light of safety and efficacy. Challenges confronting the issue hinge upon myriad studies that do not necessarily represent the heterogeneous target population of this therapeutic approach. The application of P-gp modulators has also been complicated by the promiscuous substrate-binding behaviour of P-gp, as well as toxicities related to its intrinsic presence in healthy tissue. This review capitalizes on information spanning genetics, energetics, and pharmacology, bringing to light some fundamental aspects that ought to be reconsidered in order to improve upon and design the next generation of P-gp inhibitors. PMID:27648351

  15. Disrupting P-glycoprotein function in clinical settings: what can we learn from the fundamental aspects of this transporter?

    PubMed Central

    Chung, Francisco S; Santiago, Jayson S; Jesus, Miguel Francisco M De; Trinidad, Camille V; See, Melvin Floyd E

    2016-01-01

    P-glycoprotein is one of the most well-studied drug transporters, significant for its role in cancer multiple drug resistance. However, using P-gp inhibitors with the aim of enhancing the therapeutic efficacy of anti-cancer drugs has led to disappointing outcomes. Furthermore, several lead compounds suggested by in vitro and pre-clinical studies have shown variable pharmacokinetics and therapeutic efficacies when applied in the clinical setting. This review will highlight the need to revisit a sound approach to better design and apply P-gp inhibitors in light of safety and efficacy. Challenges confronting the issue hinge upon myriad studies that do not necessarily represent the heterogeneous target population of this therapeutic approach. The application of P-gp modulators has also been complicated by the promiscuous substrate-binding behaviour of P-gp, as well as toxicities related to its intrinsic presence in healthy tissue. This review capitalizes on information spanning genetics, energetics, and pharmacology, bringing to light some fundamental aspects that ought to be reconsidered in order to improve upon and design the next generation of P-gp inhibitors.

  16. The ABC transporters in Candidatus Liberibacter asiaticus

    PubMed Central

    Li, Wenlin; Cong, Qian; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2012-01-01

    Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram-negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter-related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In-depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid-like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. PMID:22807026

  17. Is P-glycoprotein (ABCB1) a phase 0 or a phase 3 colchicine transporter depending on colchicine exposure conditions?

    SciTech Connect

    Decleves, Xavier. E-mail: xavier.decleves@univ-paris5.fr; Niel, Elisabeth; Debray, Marcel; Scherrmann, Jean-Michel

    2006-12-01

    This study investigates the P-glycoprotein (Pgp)-mediated transport of its substrates in accumulation or efflux modes under steady-state conditions. The kinetics of colchicine uptake and efflux, a substrate of both Pgp and intracellular tubulin, were studied in HL60 and HL60/DNR cells; HL60/DNR cells contain 25 times more Pgp than do HL60 cells. HL60/DNR cells in a medium containing 6.25 nM colchicine, which mimics therapeutic conditions, reached steady-state twice as rapidly as did HL60 cells, and accumulated 24-times less colchicine than did HL60 cells. The Pgp inhibitor GF120918, increased colchicine uptake by HL60 cells 1.2-fold and that of HL60/DNR cells 17-fold, while it had no effect on colchicine efflux from either cell line that had been incubated with colchicine for 24 h. Colchicine kinetics fitted well a two closed-compartment model, showing that the low intracellular accumulation of colchicine in HL60/DNR cells resulted from a 11-fold decrease in colchicine uptake and a 2.3-fold increase in colchicine efflux, that could be attributed to Pgp-mediated efflux activity in HL60/DNR cells. Intracellular colchicine was mainly and similarly distributed in the cytosol in both cell lines. These data demonstrate that the kinetics of the intracellular colchicine accumulation depend on the density of Pgp and that Pgp is more a phase 0 (preventing cellular uptake) than a phase 3 (effluxing intracellular substrate) transporter under steady-state conditions, although the situation is reversed after a short incubation time (30 min), when intracellular free colchicine concentration is probably high enough for it to be removed from the cell by Pgp.

  18. P-glycoprotein (ABCB1) transports the primary active tamoxifen metabolites endoxifen and 4-hydroxytamoxifen and restricts their brain penetration.

    PubMed

    Iusuf, Dilek; Teunissen, Sebastiaan F; Wagenaar, Els; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2011-06-01

    P-glycoprotein (P-gp, ABCB1) is a highly efficient drug efflux pump expressed in brain, liver, and small intestine, but also in tumor cells, that affects pharmacokinetics and confers therapy resistance for many anticancer drugs. The aim of this study was to investigate the impact of P-gp on tamoxifen and its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. We used in vitro transport assays and Abcb1a/1b(-/-) mice to investigate the impact of P-gp on the oral availability and brain penetration of tamoxifen and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral administration of tamoxifen (50 mg/kg) was not changed in the absence of P-gp. However, brain accumulation of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were modestly, but significantly (1.5- to 2-fold), increased. Endoxifen, however, displayed a 9-fold higher brain penetration at 4 h after administration. Endoxifen was transported by P-gp in vitro. Upon direct oral administration of endoxifen (20 mg/kg), systemic exposure was slightly decreased in Abcb1a/1b(-/-) mice, but brain accumulation of endoxifen was dramatically increased (up to 23-fold at 4 h after administration). Shortly after high-dose intravenous administration (5 or 20 mg/kg), endoxifen brain accumulation was increased only 2-fold in Abcb1a/1b(-/-) mice compared with wild-type mice, suggesting a partial saturation of P-gp at the blood-brain barrier. Endoxifen, the clinically most relevant metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo, where P-gp limits its brain penetration. P-gp might thus be relevant for tamoxifen/endoxifen resistance of P-gp-positive breast cancer and tumors positioned behind a functional blood-brain barrier. PMID:21378205

  19. Multidrug resistance-associated ABC transporters - too much of one thing, good for nothing.

    PubMed

    Prochazkova, Jirina; Lanova, Martina; Pachernik, Jiri

    2012-08-01

    Abstract Overexpression of ATP-binding cassette (ABC) transporters in cancer cells results in multidrug resistance (MDR) which leads to unsuccessful chemotherapy. The most important MDR-associated members of ABC superfamily are ABC B1/P-glycoprotein/MDR1, ABC C1/multidrug resistance associated protein 1 (MRP1), and ABC G2/BCRP. This study is not only focused on function, substrates, and localization of these popular proteins but also on other ABC C family members such as ABC C2-6/MRP2-6 and ABC C7/CFTR. Current research is mainly oriented on the cancer-promoting role of these proteins, but important lessons could also be learned from the physiological roles of these proteins or from polymorphisms affecting their function. Thorough knowledge of structure and detailed mechanism of efflux can aid in the discovery of new chemotherapy targets in the future. Although the best way on how to deal with MDR would be to prevent its development, we describe some new promising strategies on how to conquer both inherited and induced MDRs.

  20. Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo.

    PubMed

    Ahmed, Iman S; Hassan, Mariame A; Kondo, Takashi

    2015-03-01

    The administration of grapefruit juice (GFJ) has been postulated to inhibit the activity of P-glycoprotein (P-gp) transport system and thus can enhance the uptake of substrate drugs. However, for various reasons, the results obtained have been always swaying between confirmation and refutation. This study aims at re-evaluating the effect of lyophilized freshly-prepared grapefruit juice (LGFJ) prepared from the whole peeled fruit on P-gp activity using the model drug doxorubicin (DOX) in-vitro and timolol maleate (TM) in-vivo. Human uterine sarcoma MES-SA/DX5v cells, grown under nanomolar concentration of DOX and highly expressing P-gp, were used as model cells for in-vitro studies whereas white New Zealand male rabbits were used for in-vivo studies. Results showed that the accumulation of DOX in MES-SA/DX5v cells was increased by 18.3 ± 2.0% in presence of LGFJ compared to control experiments. Results from in-vivo absorption studies showed that the relative oral bioavailability of TM ingested with LGFJ was significantly higher by 70% and 43% compared to the oral bioavailability of TM ingested with saline and a commercial GFJ, respectively. This study as such confirms the inhibitory effects of LGFJ on P-gp efflux proteins and highlights the superiority of using lyophilized freshly prepared juices over the commercially available juices in research studies. Also, the results call for further studies to assess the possibility of co-administrating LGFJ with anti-cancer agents to modulate multidrug resistance in their cellular environment or incorporating LGFJ in solid dosage forms to improve oral bioavailability of drugs. PMID:24303901

  1. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  2. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  3. Inducibility of the P-glycoprotein transport activity in the marine mussel Mytilus galloprovincialis and the freshwater mussel Dreissena polymorpha.

    PubMed

    Smital, Tvrtko; Sauerborn, Roberta; Hackenberger, Branimir K

    2003-12-10

    Previous investigations directed to the determination of the P-glycoprotein (Pgp) expression in aquatic organisms have indicated the possibility of the multixenobiotic resistance mechanism (MXR) induction as a response to organic pollution. However, in numerous cases no significant and/or no clear relationship between Pgp contents and pollution level was detected. Concerning these discrepancies the results of an extensive, 3-year study of the Pgp mediated MXR induction in the selected freshwater (Dreissena polymorpha) and marine (Mytilus galloprovincialis) bivalves are presented here. The main goals of the study were to ascertain the rate-dynamic, level, as well as the possible usability of MXR in environmental biomonitoring. Since the primary result of MXR induction should be the decreased intracellular accumulation of xenobiotics, the determination of MXR induction was performed using the measurement of Pgp transport activity. We measured the accumulation or the efflux rate of the model Pgp substrate rhodamine B (RB) in gills of the mussels previously exposed to pollution. The study was performed in several steps: from the exposure experiments in laboratory, using model inducers rhodamine 123 (R123) and water extract of Diesel-2 oil (D2), to the final in situ testing in real environmental conditions. Our results confirmed that Pgp activity is induced/induces according to the level of pollution, and that 4-days period was already long enough for the significant induction and deinduction of MXR activity. However, the inducibility of Pgp transport activity was significantly limited--the maximal level of induction obtained in this study resulted in 50-60% lower RB accumulation in the gills of induced specimens (laboratory or in situ exposed to pollution), when compared to control, non-induced animals. The obtained level of Pgp related MXR induction, resulting in halfway lesser accumulation of a model pollutant (RB), extrapolated to the similar scenario with toxic

  4. ABC transporters, atherosclerosis and inflammation.

    PubMed

    Fitzgerald, Michael L; Mujawar, Zahedi; Tamehiro, Norimasa

    2010-08-01

    Atherosclerosis, driven by inflamed lipid-laden lesions, can occlude the coronary arteries and lead to myocardial infarction. This chronic disease is a major and expensive health burden. However, the body is able to mobilize and excrete cholesterol and other lipids, thus preventing atherosclerosis by a process termed reverse cholesterol transport (RCT). Insight into the mechanism of RCT has been gained by the study of two rare syndromes caused by the mutation of ABC transporter loci. In Tangier disease, loss of ABCA1 prevents cells from exporting cholesterol and phospholipid, thus resulting in the build-up of cholesterol in the peripheral tissues and a loss of circulating HDL. Consistent with HDL being an athero-protective particle, Tangier patients are more prone to develop atherosclerosis. Likewise, sitosterolemia is another inherited syndrome associated with premature atherosclerosis. Here mutations in either the ABCG5 or G8 loci, prevents hepatocytes and enterocytes from excreting cholesterol and plant sterols, including sitosterol, into the bile and intestinal lumen. Thus, ABCG5 and G8, which from a heterodimer, constitute a transporter that excretes cholesterol and dietary sterols back into the gut, while ABCA1 functions to export excess cell cholesterol and phospholipid during the biogenesis of HDL. Interestingly, a third protein, ABCG1, that has been shown to have anti-atherosclerotic activity in mice, may also act to transfer cholesterol to mature HDL particles. Here we review the relationship between the lipid transport activities of these proteins and their anti-atherosclerotic effect, particularly how they may reduce inflammatory signaling pathways. Of particular interest are recent reports that indicate both ABCA1 and ABCG1 modulate cell surface cholesterol levels and inhibit its partitioning into lipid rafts. Given lipid rafts may provide platforms for innate immune receptors to respond to inflammatory signals, it follows that loss of ABCA1 and ABCG1

  5. Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses.

    PubMed

    Kadioglu, Onat; Saeed, Mohamed E M; Valoti, Massimo; Frosini, Maria; Sgaragli, Giampietro; Efferth, Thomas

    2016-03-15

    Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis-cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean ± SD) ranging from -11.8 ± 0.54 (valspodar) to -3.98 ± 0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of -6.9 ± 0.30 kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, (3)H-1EPI, (3)H-2EPI, (14)C-1EPI, (14)C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both (3)H-2EPI and (14)C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

  6. Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses.

    PubMed

    Kadioglu, Onat; Saeed, Mohamed E M; Valoti, Massimo; Frosini, Maria; Sgaragli, Giampietro; Efferth, Thomas

    2016-03-15

    Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis-cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean ± SD) ranging from -11.8 ± 0.54 (valspodar) to -3.98 ± 0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of -6.9 ± 0.30 kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, (3)H-1EPI, (3)H-2EPI, (14)C-1EPI, (14)C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both (3)H-2EPI and (14)C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123. PMID:26807479

  7. Inward transport of [3H]-1-methyl-4-phenylpyridinium in rat isolated hepatocytes: putative involvement of a P-glycoprotein transporter.

    PubMed Central

    Martel, F.; Martins, M. J.; Hipólito-Reis, C.; Azevedo, I.

    1996-01-01

    1. The liver has an important role in the detoxification of organic cations from the circulation. [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+), a low molecular weight organic cation, is efficiently taken up and accumulated by rat hepatocytes through mechanisms partially unknown. 2. The aim of the present work was to characterize further the uptake of MPP+ by rat isolated hepatocytes. The putative interactions of a wide range of drugs, including inhibitors/substrates of P-glycoprotein, were studied. 3. The uptake of MPP+ was investigated in rat freshly isolated hepatocytes (incubated in Krebs-Henseleit medium with 200 nM [3H]-MPP+ for 5 min) and in the rat liver in situ (perfused with Krebs-Henseleit/BSA medium with 200 nM [3H]-MPP+ for 30 min). [3H]-MPP+ accumulation in the cells and in tissue was determined by liquid scintillation counting. 4. Verapamil (100 microM), quinidine (100 microM), amiloride (1 mM), (+)-tubocurarine (100 microM), vecuronium (45 microM), bilirubin (200 microM), progesterone (200 microM), daunomycin (100 microM), vinblastine (100 microM), cyclosporin A (100 microM) and cimetidine (100 microM) had a significant inhibitory effect on the accumulation of [3H]-MPP+ in isolated hepatocytes. Tetraethylammonium (100 microM) had no effect. 5. In the rat perfused liver, both cyclosporin A (100 microM) and verapamil (100 microM) had much less marked inhibitory effects as compared to their effects on isolated hepatocytes (0% against 35% and 45% against 96% of inhibition, respectively). 6. Inhibition of alkaline phosphatase activity by increasing or decreasing the pH of the incubation medium or by the presence of vanadate (1 mM) or homoarginine (500 microM) led to a significant increase in the accumulation of [3H]-MPP+ in isolated hepatocytes. 7. It was concluded that, in addition to the type I organic cation hepatic transporter, [3H]-MPP+ is taken up by rat hepatocytes through P-glycoprotein, a canalicular transport system that usually excretes

  8. Structural insights into ABC transporter mechanism

    SciTech Connect

    Oldham, Michael L.; Davidson, Amy L.; Chen, Jue

    2010-07-27

    ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

  9. Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites.

    PubMed

    Alcantara, Laura M; Kim, Junwon; Moraes, Carolina B; Franco, Caio H; Franzoi, Kathrin D; Lee, Sukjun; Freitas-Junior, Lucio H; Ayong, Lawrence S

    2013-06-01

    Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.

  10. Inhibition or knockdown of ABC transporters enhances susceptibility of adult and juvenile schistosomes to Praziquantel.

    PubMed

    Kasinathan, Ravi S; Sharma, Lalit Kumar; Cunningham, Charles; Webb, Thomas R; Greenberg, Robert M

    2014-10-01

    Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ). Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1) and other ATP binding cassette (ABC) transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR). Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3-4 weeks post infection), normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R)-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R)-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that parasite ABC

  11. On the energy-dependence of Hoechst 33342 transport by the ABC transporter LmrA.

    PubMed

    Venter, Henrietta; Velamakanni, Saroj; Balakrishnan, Lekshmy; van Veen, Hendrik W

    2008-02-15

    LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA. Furthermore, the observations on LmrA-MD raised the question whether ATP-dependent transport by LmrA in intact cells could be due to the activity of independent ABC transporters that might become upregulated in the lactococcal cells due to the overexpression of LmrA; the recently identified ABC multidrug transporter LmrCD was put forward as a possible candidate. Here, we investigated the energy coupling to the transport of the amphiphilic dye Hoechst 33342 in proteoliposomes containing purified LmrA. For this purpose, LmrA was obtained from lactococcal cells lacking the genomic lmrA and lmrCD genes, in which LmrA was expressed from a plasmid. To separate ATP-dependence from proton motive force-dependence, we also used mutant LmrA proteins, which were affected in their ability to hydrolyse ATP. Our studies in proteoliposomes demonstrate that LmrA can catalyze Hoechst 33342 transport independent of auxiliary proteins, in an ATP-dependent fashion and a transmembrane chemical proton gradient (interior acidic)-dependent fashion.

  12. Kinetic Validation of the Models for P-Glycoprotein ATP Hydrolysis and Vanadate-Induced Trapping. Proposal for Additional Steps

    PubMed Central

    Lugo, Miguel Ramón; Sharom, Frances Jane

    2014-01-01

    P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the

  13. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    PubMed

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

  14. Contribution of radixin to P-glycoprotein expression and transport activity in mouse small intestine in vivo.

    PubMed

    Yano, Kentaro; Tomono, Takumi; Sakai, Riyo; Kano, Takashi; Morimoto, Kaori; Kato, Yukio; Ogihara, Takuo

    2013-08-01

    The ERM proteins, ezrin, radixin, and moesin, are membrane-cytoskeleton cross-linkers with multiple physiological functions. We previously showed that radixin is involved in posttranslational regulation of P-glycoprotein (P-gp) in human hepatoblastoma HepG2 cells. Here, we investigated the physiological role of radixin in regulating P-gp expression and activity in the small intestine by comparing wild-type- and radixin knockout (Rdx) mice. In intestinal tissue homogenates, P-gp protein levels increased markedly from the upper part to the lower part of the small intestine in both wild-type- and Rdx(-/-) mice. In the membrane fractions, a similar pattern was seen in wild-type mice. However, the membrane expression of P-gp protein remained at the same level from the upper to the lower part of the small intestine in Rdx(-/-) mice. When rhodamine123 (Rho123), a substrate of P-gp, was orally administered to Rdx(-/-) and wild-type mice, the absorption phase of Rho123 was greater in Rdx(-/-) than in wild-type mice, whereas the elimination phase in Rdx(-/-) mice was not different from that of wild-type mice. Our results indicate that radixin plays an important role in regulating P-gp localization and P-gp functional activity at the intestinal membrane.

  15. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  16. P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

    PubMed

    Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

    2012-10-01

    We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively.

  17. Induction of expression and functional activity of P-glycoprotein efflux transporter by bioactive plant natural products.

    PubMed

    Abuznait, Alaa H; Qosa, Hisham; O'Connell, Nicholas D; Akbarian-Tefaghi, Jessica; Sylvester, Paul W; El Sayed, Khalid A; Kaddoumi, Amal

    2011-11-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug's therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3-3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31-38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. PMID:21851848

  18. Detergent-free purification of ABC (ATP-binding-cassette) transporters.

    PubMed

    Gulati, Sonali; Jamshad, Mohammed; Knowles, Timothy J; Morrison, Kerrie A; Downing, Rebecca; Cant, Natasha; Collins, Richard; Koenderink, Jan B; Ford, Robert C; Overduin, Michael; Kerr, Ian D; Dafforn, Timothy R; Rothnie, Alice J

    2014-07-15

    ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

  19. [11C]Flumazenil brain uptake is influenced by the blood-brain barrier efflux transporter P-glycoprotein

    PubMed Central

    2012-01-01

    Background [11C]Flumazenil and positron emission tomography (PET) are used clinically to assess gamma-aminobutyric acid (GABA)-ergic function and to localize epileptic foci prior to resective surgery. Enhanced P-glycoprotein (P-gp) activity has been reported in epilepsy and this may confound interpretation of clinical scans if [11C]flumazenil is a P-gp substrate. The purpose of this study was to investigate whether [11C]flumazenil is a P-gp substrate. Methods [11C]Flumazenil PET scans were performed in wild type (WT) (n = 9) and Mdr1a/1b, (the genes that encode for P-gp) double knockout (dKO) (n = 10) mice, and in naive rats (n = 10). In parallel to PET scanning, [11C]flumazenil plasma concentrations were measured in rats. For 6 of the WT and 6 of the dKO mice a second, [11C]flumazenil scan was acquired after administration of the P-gp inhibitor tariquidar. Cerebral [11C]flumazenil concentrations in WT and Mdr1a/1b dKO mice were compared (genetic disruption model). Furthermore, pre and post P-gp-blocking cerebral [11C]flumazenil concentrations were compared in all animals (pharmacological inhibition model). Results Mdr1a/1b dKO mice had approximately 70% higher [11C]flumazenil uptake in the brain than WT mice. After administration of tariquidar, cerebral [11C]flumazenil uptake in WT mice increased by about 80% in WT mice, while it remained the same in Mdr1a/1b dKO mice. In rats, cerebral [11C]flumazenil uptake increased by about 60% after tariquidar administration. Tariquidar had only a small effect on plasma clearance of flumazenil. Conclusions The present study showed that [11C]flumazenil is a P-gp substrate in rodents. Consequently, altered cerebral [11C]flumazenil uptake, as observed in epilepsy, may not reflect solely GABAA receptor density changes but also changes in P-gp activity. PMID:22455873

  20. The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms

    PubMed Central

    Natarajan, Karthika; Bhullar, Jasjeet; Shukla, Suneet; Burcu, Mehmet; Chen, Zhe-Sheng; Ambudkar, Suresh V.; Baer, Maria R.

    2013-01-01

    Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC50 of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization. PMID:23261525

  1. Effects of lipid vehicle and P-glycoprotein inhibition on the mesenteric lymphatic transport of paclitaxel in unconscious, lymph duct-cannulated rats.

    PubMed

    Cai, Qingqing; Deng, Xinxian; Li, Zhongdong; An, Dianyun; Shen, Teng; Zhong, Mingkang

    2016-01-01

    The present study examined the effects of lipid vehicle and intestinally based efflux processes on intestinal lymphatic transport of paclitaxel (PTX) in the mesenteric lymph duct-cannulated anesthetized rat model. PTX solution alone, PTX solution pretreated with the P-glycoprotein (P-gp) inhibitor verapamil and/or PTX and a 2:1 (w/w) mixture of linoleic acid:glycerol monooleate were administered intraduodenally to anesthetized rats. Coadministration of a mixture of linoleic acid-monoolein significantly increased the extent of intestinal lymphatic transport of PTX, but it had little impact on the absolute oral bioavailability of PTX. In contrast, pretreatment with verapamil increased both the extent of lymphatic transport (3.5-fold) and absolute oral bioavailability (1.8-fold). Further increase in the lymphatic transport (6.5-fold) and absolute oral bioavailability (1.8-fold) was achieved by the combination of pretreatment with verapamil and coadministration with the linoleic acid-monoolein mixture. These data indicate that the application of lipid vehicle holds promise for selectively targeted lymphatic delivery of PTX. P-gp inhibition can result in both increased intestinal lymphatic levels and absolute oral bioavailability of PTX.

  2. Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1

    PubMed Central

    Kim, Suyoung; Park, Sook-Young; Kim, Hyejeong; Kim, Dongyoung; Lee, Seon-Woo; Kim, Heung Tae; Lee, Jong-Hwan; Choi, Woobong

    2014-01-01

    Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5′-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum. PMID:25506302

  3. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids

    PubMed Central

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J.; Kwatra, Deep; Paturi, Kalyani D.; Mitra, Ashim. K.

    2010-01-01

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the cellular washing and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of PappB-A to PappA-B in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake, elevation of cyclosporine uptake in the presence of 5 μM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories. PMID:20163160

  4. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

    SciTech Connect

    Fuchs, Dominik; Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard; Naujokat, Cord

    2010-04-16

    Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

  5. Pharmacotherapy in pregnancy; effect of ABC and SLC transporters on drug transport across the placenta and fetal drug exposure.

    PubMed

    Staud, Frantisek; Cerveny, Lukas; Ceckova, Martina

    2012-11-01

    Pharmacotherapy during pregnancy is often inevitable for medical treatment of the mother, the fetus or both. The knowledge of drug transport across placenta is, therefore, an important topic to bear in mind when deciding treatment in pregnant women. Several drug transporters of the ABC and SLC families have been discovered in the placenta, such as P-glycoprotein, breast cancer resistance protein, or organic anion/cation transporters. It is thus evident that the passage of drugs across the placenta can no longer be predicted simply on the basis of their physical-chemical properties. Functional expression of placental drug transporters in the trophoblast and the possibility of drug-drug interactions must be considered to optimize pharmacotherapy during pregnancy. In this review we summarize current knowledge on the expression and function of ABC and SLC transporters in the trophoblast. Furthermore, we put this data into context with medical conditions that require maternal and/or fetal treatment during pregnancy, such as gestational diabetes, HIV infection, fetal arrhythmias and epilepsy. Proper understanding of the role of placental transporters should be of great interest not only to clinicians but also to pharmaceutical industry for future drug design and development to control the degree of fetal exposure.

  6. Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P-Glycoprotein Function in Mouse Small Intestine.

    PubMed

    Akazawa, Takanori; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2016-07-01

    The purpose of this study was to investigate whether a pharmacokinetic model integrating in vitro mdr1a efflux activity (which we previously reported) with in vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a function. In situ intestinal permeability-surface area product ratio between wild-type and mdr1a/1b (-/-) mice is one of the parameters used to describe intestinal mdr1a function. The reconstructed ratios of six mdr1a substrates (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine) and one nonsubstrate (diazepam) were consistent with the observed values reported by Adachi et al. within 2.1-fold difference. Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic modeling approach. Furthermore, we evaluated regional differences in protein expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5, abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostβ, villin1, Na(+)/K(+)-ATPase, γ-gtp) out of 46 target molecules were detected by employing our established quantitative targeted absolute proteomics technique. The protein expression amounts of mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc were highly expressed in jejunum and ileum. Mct1 and ostβ were highly expressed in ileum. The quantitative expression profiles established here should be helpful to understand and predict intestinal transporter functions. PMID:27276518

  7. The antiepileptic drug mephobarbital is not transported by P-glycoprotein or multidrug resistance protein 1 at the blood-brain barrier: a positron emission tomography study

    PubMed Central

    Mairinger, Severin; Bankstahl, Jens P.; Kuntner, Claudia; Römermann, Kerstin; Bankstahl, Marion; Wanek, Thomas; Stanek, Johann; Löscher, Wolfgang; Müller, Markus; Erker, Thomas; Langer, Oliver

    2013-01-01

    Summary Aim of this study was to determine whether the carbon-11-labelled antiepileptic drug [11C]mephobarbital is a substrate of P-glycoprotein (Pgp) and can be used to assess Pgp function at the blood-brain barrier (BBB) with positron emission tomography (PET). We performed paired PET scans in rats, wild-type (FVB) and Mdr1a/b(−/−) mice, before and after intravenous administration of the Pgp inhibitor tariquidar (15 mg/kg). Brain-to-blood AUC0-60 ratios in rats and brain AUC0-60 values of [11C]mephobarbital in wild-type and Mdr1a/b(−/−) mice were similar in scan 1 and scan 2, respectively, suggesting that in vivo brain distribution of [11C]mephobarbital is not influenced by Pgp efflux. Absence of Pgp transport was confirmed in vitro by performing concentration equilibrium transport assay in cell lines transfected with MDR1 or Mdr1a. PET experiments in wild-type mice, with and without pretreatment with the multidrug resistance protein (MRP) inhibitor MK571 (20 mg/kg), and in Mrp1(−/−) mice suggested that [11C]mephobarbital is also not transported by MRPs at the murine BBB, which was also supported by in vitro transport experiments using human MRP1-transfected cells. Our results are surprising as phenobarbital, the N-desmethyl derivative of mephobarbital, has been shown to be a substrate of Pgp, which suggests that N-methylation abolishes Pgp affinity of barbiturates. PMID:22342565

  8. Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

    PubMed Central

    2012-01-01

    Background P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. Methods Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. Results We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a

  9. P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

    PubMed

    Liu, Qian; Hou, Jinfeng; Chen, Xiaoguang; Liu, Gengtao; Zhang, Dan; Sun, Hua; Zhang, Jinlan

    2014-01-01

    FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport. PMID:25036090

  10. P-glycoprotein differentially affects escitalopram, levomilnacipran, vilazodone and vortioxetine transport at the mouse blood-brain barrier in vivo.

    PubMed

    Bundgaard, Christoffer; Eneberg, Elin; Sánchez, Connie

    2016-04-01

    P-glycoprotein (P-gp)-mediated brain efflux of xenobiotics is a well-known process, which may result in suboptimal target engagement and consequently reduced efficacy of drugs exerting their therapeutic effects in the central nervous system. In the present study the role of P-gp in transport across the blood-brain barrier (BBB) was investigated with a series of newer antidepressants (levomilnacipran, vilazodone and vortioxetine) and a control substrate (escitalopram) using P-gp knock-out (KO) and P-gp competent wild-type (WT) mice. Brain and plasma exposure time-courses were measured after an acute subcutaneous dose and at steady-state obtained after subcutaneous drug infusion by osmotic minipumps. Following acute dosing, the brain-to-plasma KO/WT exposure enhancement ratios ((AUCbrain ko/AUCplasma ko)/(AUCbrain WT/AUCplasma WT)) were 5.8 (levomilnacipran), 5.4 (vilazodone), 3.1 (escitalopram) and 0.9 (vortioxetine), respectively. At steady-state, assessment of Kp,uu (unbound brain concentrations/unbound plasma concentrations) revealed a restriction in the brain distribution in WT mice for all compounds except vortioxetine. Levomilnacipran exhibited the most pronounced efflux with a Kp,uu-value of 0.038 in WT mice which was increased to 0.37 in KO mice. Based on both the acute and steady-state distribution data, the results suggest that levomilnacipran, vilazodone and escitalopram are susceptible to P-gp mediated efflux at the BBB in vivo in mice, whereas vortioxetine was practically devoid of being affected by P-gp in vivo. The functional impact of the drug transport-controlling role of P-gp at the BBB was demonstrated by in vivo cortical serotonin transporter occupancy of vilazodone, which exhibited a 20-fold higher plasma EC50 in WT mice compared to KOs. PMID:26700248

  11. Usefulness of A Model-Based Approach for Estimating In Vitro P-Glycoprotein Inhibition Potency in a Transcellular Transport Assay.

    PubMed

    Kishimoto, Wataru; Ishiguro, Naoki; Ludwig-Schwellinger, Eva; Ebner, Thomas; Maeda, Kazuya; Sugiyama, Yuichi

    2016-02-01

    In vitro half-maximal inhibitory concentration (IC50) is a key parameter for accurately predicting the potential risk for P-glycoprotein (P-gp)--mediated drug--drug interactions. We aimed to compare the IC50 values estimated by different approaches and determine the usefulness of model-based approaches. Transcellular transport of digoxin across Caco-2 monolayer was investigated using various concentrations of P-gp inhibitors, quinidine, verapamil, and zosuquidar. To calculate IC50 values, 3 traditional parameters were used: apical-to-basal (AtoB) and basal-to-apical (BtoA) clearance (CL) with inhibitors (CLAtoB,i and CLBtoA,i) and the difference between the efflux ratios (ERs) with P-gp inhibitors (ERi) and those under complete P-gp inhibition [ER(-P-gp)]. Furthermore, a new model-based approach was applied that uses the difference between the reciprocals of CLAtoB with P-gp inhibitors (1/CLAtoB,i) and those under complete P-gp inhibition [1/CLAtoB(-P-gp)] as parameters. IC50 values obtained from 2 model-based approaches [ERi - ER(-P-gp) and 1/CLAtoB,i - 1/CLAtoB(-P-gp)] were comparable, whereas 2.6- to 6.6-fold larger IC50 values were estimated from empirical approaches (CLAtoB,i and CLBtoA,i). The reason for such difference in IC50 values is that indicators for model-based approaches, but not empirical approaches, directly reflect the P-gp function. Our new approach [1/CLAtoB,i - 1/CLAtoB(-P-gp)] based on only AtoB transcellular transport could substitute for current estimation methods using ER.

  12. Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

    PubMed

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul; Ambudkar, Suresh V

    2012-02-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  13. MDR-ABC transporters: biomarkers in rheumatoid arthritis.

    PubMed

    Márki-Zay, János; Tauberné Jakab, Katalin; Szerémy, Péter; Krajcsi, Peter

    2013-01-01

    MDR-ABC transporters are widely expressed in cell types relevant to pathogenesis of rheumatoid arthritis. Many reports demonstrate the interaction of small molecule drugs with MDR-ABC transporters. Cell-based assays for disease relevant cell types can be easily gated and could reveal specific drug targets and may increase significance and utilisation of data in clinical practice. Many commonly used DMARDs (e.g. methotrexate, sulfasalazine, leflunomide/teriflunomide, hydroxychloroquine) are ABCG2 substrates. Consequently, the activity of this transporter in patients should be determined to understand the disposition and pharmacokinetics of the therapy. In addition, MDR-ABC transporters transport a variety of endobiotics that play important roles in cell proliferation, cell migration, angiogenesis and inflammation. Therefore, MDR-ABC transporters are important biomarkers in rheumatoid arthritis. PMID:23711386

  14. Inhibition of P-glycoprotein-mediated transport by S-adenosylmethionine and cynarin in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells.

    PubMed

    Angelini, A; Di Pietro, R; Centurione, L; Castellani, M L; Conti, P; Porreca, E; Cuccurullo, F

    2012-01-01

    Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to

  15. Structural basis for the mechanism of ABC transporters.

    PubMed

    Beis, Konstantinos

    2015-10-01

    The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

  16. ABC transporter research: going strong 40 years on

    PubMed Central

    Theodoulou, Frederica L.; Kerr, Ian D.

    2015-01-01

    In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters. PMID:26517919

  17. Advances in plant-based inhibitors of P-glycoprotein.

    PubMed

    Yu, Jun; Zhou, Peng; Asenso, James; Yang, Xiao-Dan; Wang, Chun; Wei, Wei

    2016-12-01

    Multidrug resistance (MDR) has emerged as the main problem in anti-cancer therapy. Although MDR involves complex factors and processes, the main pivot is the expression of multidrug efflux pumps. P-glycoprotein (P-gp) belongs to the family of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. It functions in cellular detoxification, pumping a wide range of xenobiotic compounds out of the cell. An attractive therapeutic strategy for overcoming MDR is to inhibit the transport function of P-gp and thus, increase intracellular concentration of drugs. Recently, various types of P-gp inhibitors have been found and used in experiments. However, none of them has passed clinical trials due to their high side-effects. Hence, the search for alternatives, such as plant-based P-gp inhibitors have gained attention recently. Therefore, we give an overview of the source, function, structure and mechanism of plant-based P-gp inhibitors and give more attention to cancer-related studies. These products could be the future potential drug candidates for further research as P-gp inhibitors.

  18. Function of prokaryotic and eukaryotic ABC proteins in lipid transport.

    PubMed

    Pohl, Antje; Devaux, Philippe F; Herrmann, Andreas

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry. PMID:15749056

  19. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

    SciTech Connect

    Leslie, Elaine M.; Deeley, Roger G.; Cole, Susan P.C. . E-mail: coles@post.queensu.ca

    2005-05-01

    In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.

  20. Plant cuticular lipid export requires an ABC transporter.

    PubMed

    Pighin, Jamie A; Zheng, Huanquan; Balakshin, Laura J; Goodman, Ian P; Western, Tamara L; Jetter, Reinhard; Kunst, Ljerka; Samuels, A Lacey

    2004-10-22

    A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle.

  1. ABC transporters involved in export of cell surface glycoconjugates.

    PubMed

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-09-01

    Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.

  2. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

    Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

  3. Potent galloyl-based selective modulators targeting multidrug resistance associated protein 1 and P-glycoprotein.

    PubMed

    Pellicani, Raffaella Zoe; Stefanachi, Angela; Niso, Mauro; Carotti, Angelo; Leonetti, Francesco; Nicolotti, Orazio; Perrone, Roberto; Berardi, Francesco; Cellamare, Saverio; Colabufo, Nicola Antonio

    2012-01-12

    The multifactorial nature of chemotherapy failure in controlling cancer is often associated with the occurrence of multidrug resistance (MDR), a phenomenon likely related to the increased expression of members of the ATP binding cassette (ABC) transporter superfamily. In this respect, the most extensively characterized MDR transporters include ABCB1 (also known as MDR1 or P-glycoprotein) and ABCC1 (also known as MRP1) whose inhibition remains a priority to circumvent drug resistance. Herein, we report how the simple galloyl benzamide scaffold can be easily and properly decorated for the preparation of either MRP1 or P-gp highly selective inhibitors. In particular, some gallamides and pyrogallol-1-monomethyl ethers showed remarkable affinity and selectivity toward MRP1. On the other hand, trimethyl ether galloyl anilides, with few exceptions, exhibited moderate to very high and selective P-gp inhibition.

  4. Inhibitory Effects of Green Tea and (-)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein.

    PubMed

    Knop, Jana; Misaka, Shingen; Singer, Katrin; Hoier, Eva; Müller, Fabian; Glaeser, Hartmut; König, Jörg; Fromm, Martin F

    2015-01-01

    Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC50 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC50 values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.

  5. Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

    PubMed Central

    O’Mara, Megan L.; Mark, Alan E.

    2014-01-01

    ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations

  6. Placental ABC transporters, cellular toxicity and stress in pregnancy.

    PubMed

    Aye, Irving L M H; Keelan, Jeffrey A

    2013-04-25

    The human placenta, in addition to its roles as a nutrient transfer and endocrine organ, functions as a selective barrier to protect the fetus against the harmful effects of exogenous and endogenous toxins. Members of the ATP-binding cassette (ABC) family of transport proteins limit the entry of xenobiotics into the fetal circulation via vectorial efflux from the placenta to the maternal circulation. Several members of the ABC family, including proteins from the ABCA, ABCB, ABCC and ABCG subfamilies, have been shown to be functional in the placenta with clinically significant roles in xenobiotic efflux. However, recent findings suggest that these transporters also protect placental tissue by preventing the cellular accumulation of cytotoxic compounds such as lipids, sterols and their derivatives. Such protective functions are likely to be particularly important in pregnancies complicated by inflammatory or oxidative stress, where the generation of toxic metabolites is enhanced. For example, ABC transporters have been shown to protect against the harmful effects of hypoxia and oxidative stress through increased expression and efflux of oxysterols and glutathione conjugated xenobiotics. However, this protective capacity may be diminished in response to the same stressors. Several studies in primary human trophoblast cells and animal models have demonstrated decreased expression and activity of placental ABC transporters with inflammatory, oxidative or metabolic stress. Several clinical studies in pregnancies complicated by inflammatory conditions such as preeclampsia and gestational diabetes support these findings, although further studies are required to determine the clinical relevance of the relationships between placental ABC transporter expression and activity, and placental function in stressed pregnancies. Such studies are necessary to fully understand the consequences of pregnancy disorders on placental function and viability in order to optimise pregnancy

  7. Insights into how nucleotide-binding domains power ABC transport.

    PubMed

    Newstead, Simon; Fowler, Philip W; Bilton, Paul; Carpenter, Elisabeth P; Sadler, Peter J; Campopiano, Dominic J; Sansom, Mark S P; Iwata, So

    2009-09-01

    The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. PMID:19748342

  8. The ABCs of Candida albicans Multidrug Transporter Cdr1

    PubMed Central

    Banerjee, Atanu; Khandelwal, Nitesh Kumar; Dhamgaye, Sanjiveeni

    2015-01-01

    In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms. PMID:26407965

  9. Transepithelial transport of fusariotoxin nivalenol: mediation of secretion by ABC transporters.

    PubMed

    Tep, Jonathan; Videmann, Bernadette; Mazallon, Michèle; Balleydier, Sabine; Cavret, Séverine; Lecoeur, Sylvaine

    2007-05-15

    Mycotoxin nivalenol (NIV) is a natural contaminant of various cereal crops, animal feed and processed grains throughout the world. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. In this study, we investigated the mechanisms involved in NIV transepithelial transfer. The human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of NIV. Using metabolic inhibitors and temperature-dependent experiments, we demonstrated that basolateral-apical (BL-AP) transfer of NIV involved an energy-dependent transport whereas apical-basolateral (AP-BL) transfer was governed by passive diffusion. NIV efflux was significantly decreased in the presence of the P-glycoprotein (P-gp) inhibitor valspodar, the multi-drug resistance-associated proteins (MRPs) inhibitor MK571, but was not modified by the breast cancer resistance protein (BCRP) inhibitor Ko143. Intracellular NIV accumulation was investigated using epithelial cell lines transfected with either human P-glycoprotein or MRP2. This accumulation was significantly decreased in LLCPK1/MDR1 and MDCKII/MRP2 cells, compared to wild-type cells, and this effect was reversed by valspodar and MK571, respectively. These in vitro results suggested that NIV was a substrate for both P-glycoprotein and MRP2. This interaction may play a key role in weak intestinal absorption of NIV and the mainly predominant excretion of NIV in faeces in animal studies.

  10. Characterization of Haemonchus contortus P-glycoprotein-16 and its interaction with the macrocyclic lactone anthelmintics.

    PubMed

    Godoy, P; Che, H; Beech, R N; Prichard, R K

    2015-11-01

    Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus.

  11. Fungal ABC transporters and microbial interactions in natural environments.

    PubMed

    Schoonbeek, Henk-jan; Raaijmakers, Jos M; De Waard, Maarten A

    2002-11-01

    In natural environments, microorganisms are exposed to a wide variety of antibiotic compounds produced by competing organisms. Target organisms have evolved various mechanisms of natural resistance to these metabolites. In this study, the role of ATP-binding cassette (ABC) transporters in interactions between the plant-pathogenic fungus Botrytis cinerea and antibiotic-producing Pseudomonas bacteria was investigated in detail. We discovered that 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and phenazine-1-carboxamide (PCN), broad-spectrum antibiotics produced by Pseudomonas spp., induced expression of several ABC transporter genes in B. cinerea. Phenazines strongly induced expression of BcatrB, and deltaBcatrB mutants were significantly more sensitive to these antibiotics than their parental strain. Treatment of B. cinerea germlings with PCN strongly affected the accumulation of [14C]fludioxonil, a phenylpyrrole fungicide known to be transported by BcatrB, indicating that phenazines also are transported by BcatrB. Pseudomonas strains producing phenazines displayed a stronger antagonistic activity in vitro toward ABcatrB mutants than to the parental B. cinerea strain. On tomato leaves, phenazine-producing Pseudomonas strains were significantly more effective in reducing gray mold symptoms incited by a ABcatrB mutant than by the parental strain. We conclude that the ABC transporter BcatrB provides protection to B. cinerea in phenazine-mediated interactions with Pseudomonas spp. Collectively, these results indicate that fungal ABC transporters can play an important role in antibiotic-mediated interactions between bacteria and fungi in plant-associated environments. The implications of these findings for the implementation and sustainability of crop protection by antagonistic microorganisms are discussed. PMID:12423022

  12. Modulation of Biotransformation Systems and ABC Transporters by Benznidazole in Rats

    PubMed Central

    Perdomo, Virginia G.; Rigalli, Juan P.; Villanueva, Silvina S. M.; Ruiz, María L.; Luquita, Marcelo G.; Echenique, Claudia G.

    2013-01-01

    The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-μ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-μ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself. PMID:23877690

  13. Homology modelling of human P-glycoprotein.

    PubMed

    Domicevica, Laura; Biggin, Philip C

    2015-10-01

    P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach.

  14. Class C ABC transporters and Saccharomyces cerevisiae vacuole fusion

    PubMed Central

    Sasser, Terry L; Fratti, Rutilio A

    2014-01-01

    Membrane fusion is carried out by core machinery that is conserved throughout eukaryotes. This is comprised of Rab GTPases and their effectors, and SNARE proteins, which together are sufficient to drive the fusion of reconstituted proteoliposomes. However, an outer layer of factors that are specific to individual trafficking pathways in vivo regulates the spatial and temporal occurrence of fusion. The homotypic fusion of Saccharomyces cerevisiae vacuolar lysosomes utilizes a growing set of factors to regulate the fusion machinery that include members of the ATP binding cassette (ABC) transporter family. Yeast vacuoles have five class C ABC transporters that are known to transport a variety of toxins into the vacuole lumen as part of detoxifying the cell. We have found that ABCC transporters can also regulate vacuole fusion through novel mechanisms. For instance Ybt1 serves as negative regulator of fusion through its effects on vacuolar Ca2+ homeostasis. Additional studies showed that Ycf1 acts as a positive regulator by affecting the efficient recruitment of the SNARE Vam7. Finally, we discuss the potential interface between the translocation of lipids across the membrane bilayer, also known as lipid flipping, and the efficiency of fusion. PMID:25610719

  15. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

    PubMed Central

    Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

  16. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

    PubMed

    Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

  17. Ensemble Rule-Based Classification of Substrates of the Human ABC-Transporter ABCB1 Using Simple Physicochemical Descriptors.

    PubMed

    Demel, Michael A; Kraemer, Oliver; Ettmayer, Peter; Haaksma, Eric; Ecker, Gerhard F

    2010-03-15

    Within the last decades, the detailed knowledge on the impact of membrane bound drug efflux transporters of the ATP binding cassette (ABC) protein family on the pharmacological profile of drugs has enormously increased. Especially, ABCB1 (P-glycoprotein, P-gp, MDR1) has attracted particular interest in medicinal chemistry, since it determines the clinical efficacy, side effects and toxicity risks of drug candidates. Based on this, the development of in silico models that provide rapid and cost-effective screening tools for the classification of substrates and nonsubstrates of ABCB1 is an urgent need in contemporary ADMET profiling. A characteristic hallmark feature of this transporter is its polyspecific ligand recognition pattern. In this study we describe a method for classifying ABCB1 ligands in terms of simple, conjunctive rules (RuleFit) based on interpretable ADMET features. The retrieved results showed that models based on large, very diverse data sets gave better classification performance than models based on smaller, more homogenous training sets. The best model achieved gave a correct classification rate of 0.90 for an external validation set. Furthermore, from the interpretation of the best performing model it could be concluded that in comparison to nonsubstrates ABCB1 substrates generally show a higher number of hydrogen-bond acceptors, are more flexible and exhibit higher logP values.

  18. P-glycoprotein and its inducible expression in three bivalve species after exposure to Prorocentrum lima.

    PubMed

    Huang, Lu; Liu, Su-Li; Zheng, Jian-Wei; Li, Hong-Ye; Liu, Jie-Sheng; Yang, Wei-Dong

    2015-12-01

    P-glycoprotein (P-gp or ABCB1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms. To provide more information of P-gp in shellfish, in this study, complete cDNA of P-gp in three bivalve species including Ruditapes philippinarum, Scapharca subcrenata and Tegillarca granosa were cloned and its expressions in gill, digestive gland, adductor muscle and mantle of the three bivalves were detected after exposure to Prorocentrum lima, a toxogenic dinoflagellate. The complete sequences of R. philippinarum, S. subcrenata and T. granosa P-gp showed high homology with MDR/P-gp/ABCB proteins from other species, having a typical sequence organization as full transporters from the ABCB family. Phylogenetic analyses revealed that the amino acid sequences of P-gp from S. subcrenata and T. granosa had a closest relationship, forming an independent branch, then grouping into the other branch with Mytilus californianus, Mytilus galloprovincialis and Crassostrea gigas. However, P-gp sequences from R. philippinarum were more similar to the homologs from the more distantly related Aplysia californica than to homologs from S. subcrenata and T. granosa, suggesting that bivalves P-gp might have different paralogs. P-glycoprotein expressed in all detected tissues but there were large differences between them. After exposure to P. lima, the expression of P-gp changed in the four tissues in varying degrees within the same species and between different species, but the changes in mRNA and protein level were not always synchronous.

  19. P-glycoprotein and its inducible expression in three bivalve species after exposure to Prorocentrum lima.

    PubMed

    Huang, Lu; Liu, Su-Li; Zheng, Jian-Wei; Li, Hong-Ye; Liu, Jie-Sheng; Yang, Wei-Dong

    2015-12-01

    P-glycoprotein (P-gp or ABCB1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms. To provide more information of P-gp in shellfish, in this study, complete cDNA of P-gp in three bivalve species including Ruditapes philippinarum, Scapharca subcrenata and Tegillarca granosa were cloned and its expressions in gill, digestive gland, adductor muscle and mantle of the three bivalves were detected after exposure to Prorocentrum lima, a toxogenic dinoflagellate. The complete sequences of R. philippinarum, S. subcrenata and T. granosa P-gp showed high homology with MDR/P-gp/ABCB proteins from other species, having a typical sequence organization as full transporters from the ABCB family. Phylogenetic analyses revealed that the amino acid sequences of P-gp from S. subcrenata and T. granosa had a closest relationship, forming an independent branch, then grouping into the other branch with Mytilus californianus, Mytilus galloprovincialis and Crassostrea gigas. However, P-gp sequences from R. philippinarum were more similar to the homologs from the more distantly related Aplysia californica than to homologs from S. subcrenata and T. granosa, suggesting that bivalves P-gp might have different paralogs. P-glycoprotein expressed in all detected tissues but there were large differences between them. After exposure to P. lima, the expression of P-gp changed in the four tissues in varying degrees within the same species and between different species, but the changes in mRNA and protein level were not always synchronous. PMID:26539802

  20. Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.

    PubMed

    Coleman, Jonathan A; Quazi, Faraz; Molday, Robert S

    2013-03-01

    Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

  1. A novel application of t-statistics to objectively assess the quality of IC50 fits for P-glycoprotein and other transporters.

    PubMed

    O'Connor, Michael; Lee, Caroline; Ellens, Harma; Bentz, Joe

    2015-02-01

    Current USFDA and EMA guidance for drug transporter interactions is dependent on IC50 measurements as these are utilized in determining whether a clinical interaction study is warranted. It is therefore important not only to standardize transport inhibition assay systems but also to develop uniform statistical criteria with associated probability statements for generation of robust IC50 values, which can be easily adopted across the industry. The current work provides a quantitative examination of critical factors affecting the quality of IC50 fits for P-gp inhibition through simulations of perfect data with randomly added error as commonly observed in the large data set collected by the P-gp IC50 initiative. The types of errors simulated were (1) variability in replicate measures of transport activity; (2) transformations of error-contaminated transport activity data prior to IC50 fitting (such as performed when determining an IC50 for inhibition of P-gp based on efflux ratio); and (3) the lack of well defined "no inhibition" and "complete inhibition" plateaus. The effect of the algorithm used in fitting the inhibition curve (e.g., two or three parameter fits) was also investigated. These simulations provide strong quantitative support for the recommendations provided in Bentz et al. (2013) for the determination of IC50 values for P-gp and demonstrate the adverse effect of data transformation prior to fitting. Furthermore, the simulations validate uniform statistical criteria for robust IC50 fits in general, which can be easily implemented across the industry. A calibration of the t-statistic is provided through calculation of confidence intervals associated with the t-statistic. PMID:25692007

  2. The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance.

    PubMed

    Dermauw, Wannes; Van Leeuwen, Thomas

    2014-02-01

    About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.

  3. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  4. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  5. Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein

    PubMed Central

    Weidner, Lora D.; Fung, King Leung; Kannan, Pavitra; Moen, Janna K.; Kumar, Jeyan S.; Mulder, Jan; Innis, Robert B.; Gottesman, Michael M.

    2016-01-01

    Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [11C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes. PMID:26658428

  6. Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein.

    PubMed

    Weidner, Lora D; Fung, King Leung; Kannan, Pavitra; Moen, Janna K; Kumar, Jeyan S; Mulder, Jan; Innis, Robert B; Gottesman, Michael M; Hall, Matthew D

    2016-02-01

    Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [(11)C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.

  7. P-glycoprotein activity and biological response

    SciTech Connect

    Vaalburg, W. . E-mail: w.vaalburg@pet.umcg.nl; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-09-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators.

  8. Grapefruit juice-drug interactions: Grapefruit juice and its components inhibit P-glycoprotein (ABCB1) mediated transport of talinolol in Caco-2 cells.

    PubMed

    de Castro, Whocely Victor; Mertens-Talcott, Susanne; Derendorf, Hartmut; Butterweck, Veronika

    2007-10-01

    To investigate the potential interaction between selected ingredients of grapefruit juice and, the transport of talinolol, a P-gp substrate, across Caco-2 cells monolayers was determined in the absence and presence of distinct concentrations of grapefruit juice, bergamottin, 6',7'-dihydroxybergamottin, 6',7'-epoxybergamottin, naringin, and naringenin. Talinolol permeability was selectively inhibited by grapefruit juice and its components. The furano coumarin, 6',7'-epoxybergamottin, was the most potent inhibitor (IC(50) = 0.7 microM), followed by 6',7'-dihydroxybergamottin (IC(50) = 34 microM) and bergamottin that did not show any inhibition at concentrations up to 10 microM. The flavonoid aglycone naringenin was around 10-fold more potent than its glycoside naringin with IC(50) values of 236 and 2409 microM, respectively. The flavonoids and furanocoumarins tested in this study are in the same range of concentration they are present in the juice contributing, therefore, for the overall inhibitory effect of GFJ on P-gp activity. The in vitro data suggest that compounds present in grapefruit juice are able to inhibit the P-gp activity modifying the disposition of drugs that are P-gp substrates such as talinolol. PMID:17542018

  9. P-glycoprotein is a major determinant of norbuprenorphine brain exposure and antinociception.

    PubMed

    Brown, Sarah M; Campbell, Scott D; Crafford, Amanda; Regina, Karen J; Holtzman, Michael J; Kharasch, Evan D

    2012-10-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(-/-) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(-/-) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(-/-) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID:22739506

  10. P-Glycoprotein Is a Major Determinant of Norbuprenorphine Brain Exposure and Antinociception

    PubMed Central

    Brown, Sarah M.; Campbell, Scott D.; Crafford, Amanda; Regina, Karen J.; Holtzman, Michael J.

    2012-01-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(−/−) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(−/−) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(−/−) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID

  11. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

    PubMed

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W

    2013-02-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

  12. Modulation of Expression and Activity of ABC Transporters by the Phytoestrogen Genistein. Impact on Drug Disposition.

    PubMed

    Rigalli, Juan Pablo; Ciriaci, Nadia; Mottino, Aldo Domingo; Catania, Viviana Alicia; Ruiz, María Laura

    2016-01-01

    ATP binding cassette (ABC) transporters are involved in drug absorption, distribution and elimination. They also mediate multidrug resistance in cancer cells. Isoflavones, such as genistein (GNT), belong to a class of naturally-occurring compounds found at high concentrations in commonly consumed soya based-foods and dietary supplements. GNT and its metabolites interact with ABC transporters as substrates, inhibitors and/or modulators of their expression. This review compiles information about regulation of ABC transporters by GNT with special emphasis on the three major groups of ABC transporters involved in excretion of endo- and xenobiotics as follows: Pglycoprotein (MDR1, ABCB1), a group of multidrug resistance associated proteins (MRPs, ABCC subfamily) and ABCG2 (BCRP), an ABC half-transporter. The impact of these regulations on potential GNT-drug interactions is further considered. PMID:27048380

  13. Enhancing Activity of Anticancer Drugs in Multidrug Resistant Tumors by Modulating P-Glycoprotein through Dietary Nutraceuticals.

    PubMed

    Khan, Muhammad; Maryam, Amara; Mehmood, Tahir; Zhang, Yaofang; Ma, Tonghui

    2015-01-01

    Multidrug resistance is a principal mechanism by which tumors become resistant to structurally and functionally unrelated anticancer drugs. Resistance to chemotherapy has been correlated with overexpression of p-glycoprotein (p-gp), a member of the ATP-binding cassette (ABC) superfamily of membrane transporters. P-gp mediates resistance to a broad-spectrum of anticancer drugs including doxorubicin, taxol, and vinca alkaloids by actively expelling the drugs from cells. Use of specific inhibitors/blocker of p-gp in combination with clinically important anticancer drugs has emerged as a new paradigm for overcoming multidrug resistance. The aim of this paper is to review p-gp regulation by dietary nutraceuticals and to correlate this dietary nutraceutical induced-modulation of p-gp with activity of anticancer drugs. PMID:26514453

  14. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

  15. Screening of Streptococcus pneumoniae ABC transporter mutants demonstrates that LivJHMGF, a branched-chain amino acid ABC transporter, is necessary for disease pathogenesis.

    PubMed

    Basavanna, Shilpa; Khandavilli, Suneeta; Yuste, Jose; Cohen, Jonathan M; Hosie, Arthur H F; Webb, Alexander J; Thomas, Gavin H; Brown, Jeremy S

    2009-08-01

    Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (DeltalivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the DeltalivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The DeltalivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens. PMID:19470745

  16. Identification of ABC Transporter Genes of Fusarium graminearum with Roles in Azole Tolerance and/or Virulence

    PubMed Central

    Döll, Katharina; Karlovsky, Petr; Deising, Holger B.; Wirsel, Stefan G. R.

    2013-01-01

    Fusarium graminearum is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of F. graminearum representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of FgABC3 and FgABC4 belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of F. graminearum revealed that, irrespective of their chemotypes, deletion mutants of FgABC1 (ABC-C subfamily group V) and FgABC3 were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that FgABC3 may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, FgABC1 may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of F. graminearum. PMID:24244413

  17. Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

    PubMed Central

    Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Kumar, Pradeep; Tyagi, Charu; du Toit, Lisa C.; Pillay, Viness

    2015-01-01

    The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

  18. Transcription factors that mediate epithelial–mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

    PubMed Central

    Saxena, M; Stephens, M A; Pathak, H; Rangarajan, A

    2011-01-01

    Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial–mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance. PMID:21734725

  19. Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii.

    PubMed Central

    Wanner, C; Soppa, J

    1999-01-01

    More than 40 nitrate respiration-deficient mutants of Haloferax volcanii belonging to three different phenotypic classes were isolated. All 15 mutants of the null phenotype were complemented with a genomic library of the wild type. Wild-type copies of mutated genes were recovered from complemented mutants using two different approaches. The DNA sequences of 13 isolated fragments were determined. Five fragments were found to overlap; therefore nine different genomic regions containing genes essential for nitrate respiration could be identified. Three genomic regions containing genes coding for subunits of ABC transporters were further characterized. In two cases, genes coding for an ATP-binding subunit and a permease subunit were clustered and overlapped by four nucleotides. The third gene for a permease subunit had no additional ABC transporter gene in proximity. One ABC transporter was found to be glucose specific. The mutant reveals that the ABC transporter solely mediates anaerobic glucose transport. Based on sequence similarity, the second ABC transporter is proposed to be molybdate specific, explaining its essential role in nitrate respiration. The third ABC transporter is proposed to be anion specific. Genome sequencing has shown that ABC transporters are widespread in Archaea. Nevertheless, this study represents only the second example of a functional characterization. PMID:10430572

  20. Nitrite Transport Activity of the ABC-Type Cyanate Transporter of the Cyanobacterium Synechococcus elongatus▿

    PubMed Central

    Maeda, Shin-ichi; Omata, Tatsuo

    2009-01-01

    In addition to the ATP-binding cassette (ABC)-type nitrate/nitrite-bispecific transporter, which has a high affinity for both substrates (Km, ∼1 μM), Synechococcus elongatus has an active nitrite transport system with an apparent Km (NO2−) value of 20 μM. We found that this activity depends on the cynABD genes, which encode a putative cyanate (NCO−) ABC-type transporter. Accordingly, nitrite transport by CynABD was competitively inhibited by NCO− with a Ki value of 0.025 μM. The transporter was induced under conditions of nitrogen deficiency, and the induced cells showed a Vmax value of 11 to 13 μmol/mg of chlorophyll per h for cyanate or nitrite, which could supply ∼30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and regulation at transcriptional and posttranslational levels suggested that the physiological role of the bispecific cyanate/nitrite transporter in S. elongatus is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in the medium. Its contribution to nitrite assimilation was significant in a mutant lacking the ABC-type nitrate/nitrite transporter, suggesting a possible role for CynABD in nitrite assimilation by cyanobacterial species that lack another high-affinity mechanism(s) for nitrite transport. PMID:19286804

  1. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  2. Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

    PubMed

    Qosa, Hisham; Lichter, Jessica; Sarlo, Mark; Markandaiah, Shashirekha S; McAvoy, Kevin; Richard, Jean-Philippe; Jablonski, Michael R; Maragakis, Nicholas J; Pasinelli, Piera; Trotti, Davide

    2016-08-01

    The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313.

  3. Research Progress on the Role of ABC Transporters in the Drug Resistance Mechanism of Intractable Epilepsy

    PubMed Central

    Xiong, Jie; Mao, Ding-an; Liu, Li-qun

    2015-01-01

    The pathogenesis of intractable epilepsy is not fully clear. In recent years, both animal and clinical trials have shown that the expression of ATP-binding cassette (ABC) transporters is increased in patients with intractable epilepsy; additionally, epileptic seizures can lead to an increase in the number of sites that express ABC transporters. These findings suggest that ABC transporters play an important role in the drug resistance mechanism of epilepsy. ABC transporters can perform the funcions of a drug efflux pump, which can reduce the effective drug concentration at epilepsy lesions by reducing the permeability of the blood brain barrier to antiepileptic drugs, thus causing resistance to antiepileptic drugs. Given the important role of ABC transporters in refractory epilepsy drug resistance, antiepileptic drugs that are not substrates of ABC transporters were used to obtain ABC transporter inhibitors with strong specificity, high safety, and few side effects, making them suitable for long-term use; therefore, these drugs can be used for future clinical treatment of intractable epilepsy. PMID:26491660

  4. Characterization of Two ABC Transporters from Biocontrol and Phytopathogenic Fusarium oxysporus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ABC transporter genes from four strains of Fusarium oxysporum [two biocontrol and two phytopathogenic (f. sp. lycopersici Race 1) isolates] indicated that this gene is well conserved. However, sequences of promoter regions of FoABC1 differed between 8 phytopathogenic and 11 biocontrol strains of F....

  5. Plant ABC Transporters Enable Many Unique Aspects of a Terrestrial Plant's Lifestyle.

    PubMed

    Hwang, Jae-Ung; Song, Won-Yong; Hong, Daewoong; Ko, Donghwi; Yamaoka, Yasuyo; Jang, Sunghoon; Yim, Sojeong; Lee, Eunjung; Khare, Deepa; Kim, Kyungyoon; Palmgren, Michael; Yoon, Hwan Su; Martinoia, Enrico; Lee, Youngsook

    2016-03-01

    Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant's lifestyle, by transporting various compounds across specific membranes of the plant. PMID:26902186

  6. Equilibrated Atomic Models of Outward-Facing P-glycoprotein and Effect of ATP Binding on Structural Dynamics

    PubMed Central

    Pan, Lurong; Aller, Stephen G.

    2015-01-01

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms. PMID:25600711

  7. Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.

    PubMed

    Pan, Lurong; Aller, Stephen G

    2015-01-20

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

  8. On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells.

    PubMed

    Seeland, Swen; Treiber, Alexander; Hafner, Mathias; Huwyler, Jörg

    2011-07-01

    The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the

  9. New insight into p-glycoprotein as a drug target.

    PubMed

    Breier, Albert; Gibalova, Lenka; Seres, Mario; Barancik, Miroslav; Sulova, Zdenka

    2013-01-01

    Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce

  10. New insight into p-glycoprotein as a drug target.

    PubMed

    Breier, Albert; Gibalova, Lenka; Seres, Mario; Barancik, Miroslav; Sulova, Zdenka

    2013-01-01

    Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce

  11. Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

    PubMed

    Manjula, M; Pampa, K J; Kumar, S M; Mukherjee, S; Kunishima, N; Rangappa, K S; Lokanath, N K

    2015-03-27

    The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

  12. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay ® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  13. 3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.

    PubMed

    Fribourg, Pierre Frederic; Chami, Mohamed; Sorzano, Carlos Oscar S; Gubellini, Francesca; Marabini, Roberto; Marco, Sergio; Jault, Jean-Michel; Lévy, Daniel

    2014-05-15

    ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.

  14. GintABC1 encodes a putative ABC transporter of the MRP subfamily induced by Cu, Cd, and oxidative stress in Glomus intraradices.

    PubMed

    González-Guerrero, Manuel; Benabdellah, Karim; Valderas, Ascensión; Azcón-Aguilar, Concepción; Ferrol, Nuria

    2010-02-01

    A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two six-transmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD)(2) topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra- vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.

  15. Role of ABC and Solute Carrier Transporters in the Placental Transport of Lamivudine.

    PubMed

    Ceckova, Martina; Reznicek, Josef; Ptackova, Zuzana; Cerveny, Lukas; Müller, Fabian; Kacerovsky, Marian; Fromm, Martin F; Glazier, Jocelyn D; Staud, Frantisek

    2016-09-01

    Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport. PMID:27401571

  16. Control of Plasma Membrane Permeability by ABC Transporters

    PubMed Central

    Khakhina, Svetlana; Johnson, Soraya S.; Manoharlal, Raman; Russo, Sarah B.; Blugeon, Corinne; Lemoine, Sophie; Sunshine, Anna B.; Dunham, Maitreya J.; Cowart, L. Ashley; Devaux, Frédéric

    2014-01-01

    ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. PMID:25724885

  17. Multidrug-Resistance Transporter AbcA Secretes Staphylococcus aureus Cytolytic Toxins.

    PubMed

    Yoshikai, Hirono; Kizaki, Hayato; Saito, Yuki; Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2016-01-15

    Phenol-soluble modulins (PSMs) are Staphylococcus aureus cytolytic toxins that lyse erythrocytes and neutrophils and have important functions in the S. aureus infectious process. The molecular mechanisms of PSM secretion, however, are not well understood. Here we report that knockout of the multidrug-resistance ABC transporter AbcA, which contributes to S. aureus resistance against antibiotics and chemicals, diminished the secreted amount of PSM, leading to the accumulation of PSM in the intracellular fraction. The amount of PSM in the culture supernatants of the abcA knockout mutants was restored by introduction of the wild-type abcA gene, whereas it was not completely restored by introduction of mutant abcA genes encoding AbcA mutant proteins carrying amino acid substitutions in the adenosine triphosphate binding motifs. The abcA knockout mutant exhibited attenuated virulence in a mouse systemic infection model. These findings suggest that the multidrug resistance transporter AbcA secretes PSMs and contributes to S. aureus virulence.

  18. Multidrug-Resistance Transporter AbcA Secretes Staphylococcus aureus Cytolytic Toxins.

    PubMed

    Yoshikai, Hirono; Kizaki, Hayato; Saito, Yuki; Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2016-01-15

    Phenol-soluble modulins (PSMs) are Staphylococcus aureus cytolytic toxins that lyse erythrocytes and neutrophils and have important functions in the S. aureus infectious process. The molecular mechanisms of PSM secretion, however, are not well understood. Here we report that knockout of the multidrug-resistance ABC transporter AbcA, which contributes to S. aureus resistance against antibiotics and chemicals, diminished the secreted amount of PSM, leading to the accumulation of PSM in the intracellular fraction. The amount of PSM in the culture supernatants of the abcA knockout mutants was restored by introduction of the wild-type abcA gene, whereas it was not completely restored by introduction of mutant abcA genes encoding AbcA mutant proteins carrying amino acid substitutions in the adenosine triphosphate binding motifs. The abcA knockout mutant exhibited attenuated virulence in a mouse systemic infection model. These findings suggest that the multidrug resistance transporter AbcA secretes PSMs and contributes to S. aureus virulence. PMID:26160745

  19. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. PMID:26944019

  20. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.

  1. P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins.

    PubMed

    Huang, Lu; Wang, Jie; Chen, Wen-Chang; Li, Hong-Ye; Liu, Jie-Sheng; Tao Jiang; Yang, Wei-Dong

    2014-08-01

    Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins. PMID:24811006

  2. The Predicted ABC Transporter AbcEDCBA Is Required for Type IV Secretion System Expression and Lysosomal Evasion by Brucella ovis

    PubMed Central

    Silva, Teane M. A.; Mol, Juliana P. S.; Winter, Maria G.; Atluri, Vidya; Xavier, Mariana N.; Pires, Simone F.; Paixão, Tatiane A.; Andrade, Hélida M.; Santos, Renato L.; Tsolis, Renee M.

    2014-01-01

    Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporterabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells. PMID:25474545

  3. Transcriptome-Based Identification of ABC Transporters in the Western Tarnished Plant Bug Lygus hesperus

    PubMed Central

    Hull, J. Joe; Chaney, Kendrick; Geib, Scott M.; Fabrick, Jeffrey A.; Brent, Colin S.; Walsh, Douglas; Lavine, Laura Corley

    2014-01-01

    ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences) from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus), a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species. PMID:25401762

  4. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  5. Computational classification models for predicting the interaction of drugs with P-glycoprotein and breast cancer resistance protein.

    PubMed

    Erić, S; Kalinić, M; Ilić, K; Zloh, M

    2014-01-01

    P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) are two members of the adenosine triphosphate (ATP) binding cassette (ABC) family of transporters which function as membrane efflux transporters and display considerable substrate promiscuity. Both are known to significantly influence the absorption, distribution and elimination of drugs, mediate drug-drug interactions and contribute to multiple drug resistance (MDR) of cancer cells. Correspondingly, timely characterization of the interaction of novel leads and drug candidates with these two transporters is of great importance. In this study, several computational classification models for prediction of transport and inhibition of P-gp and BCRP, respectively, were developed based on newly compiled and critically evaluated experimental data. Artificial neural network (ANN) and support vector machine (SVM) ensemble based models were explored, as well as knowledge-based approaches to descriptor selection. The average overall classification accuracy of best performing models was 82% for P-gp transport, 88% for BCRP transport, 89% for P-gp inhibition and 87% for BCRP inhibition, determined across an array of different test sets. An analysis of substrate overlap between P-gp and BCRP was also performed. The accuracy, simplicity and interpretability of the proposed models suggest that they could be of significant utility in the drug discovery and development settings.

  6. A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

    PubMed

    Chen, Lin; Duan, Kangmin

    2016-05-01

    Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell. PMID:26953208

  7. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

    PubMed

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J; Howard, Julie; Wei, Shen L; van Veen, Hendrik W

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  8. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled

    PubMed Central

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J.; Howard, Julie; Wei, Shen L.; van Veen, Hendrik W.

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  9. Single liposome analysis of peptide translocation by the ABC transporter TAPL

    PubMed Central

    Zollmann, Tina; Moiset, Gemma; Tumulka, Franz; Tampé, Robert; Poolman, Bert; Abele, Rupert

    2015-01-01

    ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters. PMID:25646430

  10. Transcriptome-based identification of ABC transporters in the western tarnished plant bug lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic cle...

  11. Natural Products based P-glycoprotein Activators for Improved β-amyloid Clearance in Alzheimer's Disease: An in silico Approach.

    PubMed

    Shinde, Pravin; Vidyasagar, Nikhil; Dhulap, Sivakami; Dhulap, Abhijeet; Hirwani, Raj

    2015-01-01

    Alzheimer's disease is an age related disorder and is defined to be progressive, irreversible neurodegenerative disease. The potential targets which are associated with the Alzheimer's disease are cholinesterases, N-methyl-D-aspartate receptor, Beta secretase 1, Pregnane X receptor (PXR) and P-glycoprotein (Pgp). P-glycoprotein is a member of the ATP binding cassette (ABC) transporter family, which is an important integral of the blood-brain, blood-cerebrospinal fluid and the blood-testis barrier. Reports from the literature provide evidences that the up-regulation of the efflux pump is liable for a decrease in β -amyloid intracellular accumulation and is an important hallmark in Alzheimer's disease (AD). Thus, targeting β-amyloid clearance by stimulating Pgp could be a useful strategy to prevent Alzheimer's advancement. Currently available drugs provide limited effectiveness and do not assure to cure Alzheimer's disease completely. On the other hand, the current research is now directed towards the development of synthetic or natural based therapeutics which can delay the onset or progression of Alzheimer's disease. Since ancient time medicinal plants such as Withania somnifera, Bacopa monieri, Nerium indicum have been used to prevent neurological disorders including Alzheimer's disease. Till today around 125 Indian medicinal plants have been screened on the basis of ethnopharmacology for their activity against neurological disorders. In this paper, we report bioactives from natural sources which show binding affinity towards the Pgp receptor using ligand based pharmacophore development, virtual screening, molecular docking and molecular dynamics simulation studies for the bioactives possessing acceptable ADME properties. These bioactives can thus be useful to treat Alzheimer's disease.

  12. P-glycoprotein alters blood–brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

    PubMed Central

    Ma, Aimei; Wang, Cuicui; Chen, Yinghui; Yuan, Weien

    2013-01-01

    P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood–brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy. PMID:24348021

  13. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  14. Tetrandrine and fangchinoline, bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells.

    PubMed

    Sun, Yan Fang; Wink, Michael

    2014-01-01

    The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy. PMID:24856768

  15. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies.

    PubMed

    Li, Wen; Zhang, Han; Assaraf, Yehuda G; Zhao, Kun; Xu, Xiaojun; Xie, Jinbing; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-07-01

    Multidrug resistance is a key determinant of cancer chemotherapy failure. One of the major causes of multidrug resistance is the enhanced efflux of drugs by membrane ABC transporters. Targeting ABC transporters projects a promising approach to eliminating or suppressing drug resistance in cancer treatment. To reveal the functional mechanisms of ABC transporters in drug resistance, extensive studies have been conducted from identifying drug binding sites to elucidating structural dynamics. In this review article, we examined the recent crystal structures of ABC proteins to depict the functionally important structural elements, such as domains, conserved motifs, and critical amino acids that are involved in ATP-binding and drug efflux. We inspected the drug-binding sites on ABC proteins and the molecular mechanisms of various substrate interactions with the drug binding pocket. While our continuous battle against drug resistance is far from over, new approaches and technologies have emerged to push forward our frontier. Most recent developments in anti-MDR strategies include P-gp inhibitors, RNA-interference, nano-medicines, and delivering combination strategies. With the advent of the 'Omics' era - genomics, epigenomics, transcriptomics, proteomics, and metabolomics - these disciplines play an important role in fighting the battle against chemoresistance by further unraveling the molecular mechanisms of drug resistance and shed light on medical therapies that specifically target MDR. PMID:27449595

  16. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies.

    PubMed

    Li, Wen; Zhang, Han; Assaraf, Yehuda G; Zhao, Kun; Xu, Xiaojun; Xie, Jinbing; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-07-01

    Multidrug resistance is a key determinant of cancer chemotherapy failure. One of the major causes of multidrug resistance is the enhanced efflux of drugs by membrane ABC transporters. Targeting ABC transporters projects a promising approach to eliminating or suppressing drug resistance in cancer treatment. To reveal the functional mechanisms of ABC transporters in drug resistance, extensive studies have been conducted from identifying drug binding sites to elucidating structural dynamics. In this review article, we examined the recent crystal structures of ABC proteins to depict the functionally important structural elements, such as domains, conserved motifs, and critical amino acids that are involved in ATP-binding and drug efflux. We inspected the drug-binding sites on ABC proteins and the molecular mechanisms of various substrate interactions with the drug binding pocket. While our continuous battle against drug resistance is far from over, new approaches and technologies have emerged to push forward our frontier. Most recent developments in anti-MDR strategies include P-gp inhibitors, RNA-interference, nano-medicines, and delivering combination strategies. With the advent of the 'Omics' era - genomics, epigenomics, transcriptomics, proteomics, and metabolomics - these disciplines play an important role in fighting the battle against chemoresistance by further unraveling the molecular mechanisms of drug resistance and shed light on medical therapies that specifically target MDR.

  17. Watching conformational dynamics of ABC transporters with single-molecule tools.

    PubMed

    Husada, Florence; Gouridis, Giorgos; Vietrov, Ruslan; Schuurman-Wolters, Gea K; Ploetz, Evelyn; de Boer, Marijn; Poolman, Bert; Cordes, Thorben

    2015-10-01

    ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

  18. An ABC pleiotropic drug resistance transporter of Fusarium graminearum with a role in crown and root diseases of wheat.

    PubMed

    Gardiner, Donald M; Stephens, Amber E; Munn, Alan L; Manners, John M

    2013-11-01

    FgABC1 (FGSG_04580) is predicted to encode a pleiotropic drug resistance class ABC transporter in Fusarium graminearum, a globally important pathogen of wheat. Deletion mutants of FgABC1 showed reduced virulence towards wheat in crown and root infection assays but were unaltered in infectivity on barley. Expression of FgABC1 during head blight and crown rot disease increases during the necrotrophic phases of infection suggestive of a role for FgABC1 in late infection stages in different tissue types. Deletion of FgABC1 also led to increased sensitivity of the fungus to the antifungal compound benalaxyl in culture, but the response to known cereal defence compounds, gramine, 2-benzoxazalinone and tryptamine was unaltered. FgABC1 appears to have a role in protecting the fungus from antifungal compounds and is likely to help combat as yet unidentified wheat defence compounds during disease development.

  19. The role of ABCG-type ABC transporters in phytohormone transport

    PubMed Central

    Borghi, Lorenzo; Kang, Joohyun; Ko, Donghwi; Lee, Youngsook; Martinoia, Enrico

    2015-01-01

    Plant hormones (phytohormones) integrate endogenous and exogenous signals thus synchronizing plant growth with environmental and developmental changes. Similar to animals, phytohormones have distinct source and target tissues, hence controlled transport and focused targeting are required for their functions. Many evidences accumulated in the last years about the regulation of long-distance and directional transport of phytohormones. ATP-binding cassette (ABC) transporters turned out to play major roles in routing phytohormones not only in the plant body but also towards the outer environment. The ABCG-type proteins ABCG25 and ABCG40 are high affinity abscisic acid (ABA) transporters. ABCG14 is highly co-expressed with cytokinin biosynthesis and is the major root-to-shoot cytokinin transporter. Pleiotropic drug resistance1 (PDR1) from Petunia hybrida transports strigolactones (SLs) from the root tip to the plant shoot but also outside to the rhizosphere, where SLs are the main attractants to mycorrhizal fungi. Last but not least, ABCG36 and ABCG37 possibly play a dual role in coumarine and IBA transport. PMID:26517905

  20. Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae*

    PubMed Central

    Gupta, Rakeshkumar P.; Kueppers, Petra; Hanekop, Nils; Schmitt, Lutz

    2014-01-01

    Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters. PMID:24733388

  1. Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2015-10-01

    The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABC subfamily B (ABCB) display very high substrate specificity compared with their mammalian counterparts that are often associated with multi-drug resistance phenomena. In this review, we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plant Arabidopsis reveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins that chaperon both transporters to the plasma membrane in an action that seems to involve heat shock protein (Hsp)90. Further, both transporters are phosphorylated at regulatory domains that connect both nt-binding folds. Taken together, it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI). PMID:26517911

  2. How much do we know about drug handling by SLC and ABC drug transporters in children?

    PubMed

    Nigam, S K; Bhatnagar, V

    2013-07-01

    Although solute carrier (SLC) and ATP-binding cassette (ABC) transporters are critical to the absorption, distribution, and elimination of many small-molecule drugs in children, how these transporters regulate pediatric drug handling remains unclear. For proper dosing and to diminish toxicity, we need a better understanding of how organ development and functional maturation, as well as developmental changes in systemic physiology, impact transporter-mediated drug handling at pediatric developmental stages from the preterm infant through adolescence.

  3. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line.

    PubMed

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong; Li, Yunman; Huang, Wenlong

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein.

  4. Blood-brain barrier P-glycoprotein function in Alzheimer's disease.

    PubMed

    van Assema, Daniëlle M E; Lubberink, Mark; Bauer, Martin; van der Flier, Wiesje M; Schuit, Robert C; Windhorst, Albert D; Comans, Emile F I; Hoetjes, Nikie J; Tolboom, Nelleke; Langer, Oliver; Müller, Markus; Scheltens, Philip; Lammertsma, Adriaan A; van Berckel, Bart N M

    2012-01-01

    A major pathological hallmark of Alzheimer's disease is accumulation of amyloid-β in senile plaques in the brain. Evidence is accumulating that decreased clearance of amyloid-β from the brain may lead to these elevated amyloid-β levels. One of the clearance pathways of amyloid-β is transport across the blood-brain barrier via efflux transporters. P-glycoprotein, an efflux pump highly expressed at the endothelial cells of the blood-brain barrier, has been shown to transport amyloid-β. P-glycoprotein function can be assessed in vivo using (R)-[(11)C]verapamil and positron emission tomography. The aim of this study was to assess blood-brain barrier P-glycoprotein function in patients with Alzheimer's disease compared with age-matched healthy controls using (R)-[(11)C]verapamil and positron emission tomography. In 13 patients with Alzheimer's disease (age 65 ± 7 years, Mini-Mental State Examination 23 ± 3), global (R)-[(11)C]verapamil binding potential values were increased significantly (P = 0.001) compared with 14 healthy controls (aged 62 ± 4 years, Mini-Mental State Examination 30 ± 1). Global (R)-[(11)C]verapamil binding potential values were 2.18 ± 0.25 for patients with Alzheimer's disease and 1.77 ± 0.41 for healthy controls. In patients with Alzheimer's disease, higher (R)-[(11)C]verapamil binding potential values were found for frontal, parietal, temporal and occipital cortices, and posterior and anterior cingulate. No significant differences between groups were found for medial temporal lobe and cerebellum. These data show altered kinetics of (R)-[(11)C]verapamil in Alzheimer's disease, similar to alterations seen in studies where P-glycoprotein is blocked by a pharmacological agent. As such, these data indicate that P-glycoprotein function is decreased in patients with Alzheimer's disease. This is the first direct evidence that the P-glycoprotein transporter at the blood-brain barrier is compromised in sporadic

  5. A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi which acts as a disease virulence factor, aiding fungal pathogenesis of cereals spikelets and spread of the economically important Fusarium head blight (FHB) disease. Previously, a fragment of a wheat ABC transporter gene was shown to be...

  6. Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.

    PubMed

    Gutay-Tóth, Zsuzsanna; Fenyvesi, Ferenc; Bársony, Orsolya; Szente, Lajos; Goda, Katalin; Szabó, Gábor; Bacsó, Zsolt

    2016-03-01

    The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.

  7. Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells.

    PubMed

    Hakkarainen, Jenni J; Rilla, Kirsi; Suhonen, Marjukka; Ruponen, Marika; Forsberg, Markus M

    2014-03-01

    1.  Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2.  BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3.  P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4.  In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates.

  8. Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of the Phytophagous Leaf Beetle Chrysomela populi

    PubMed Central

    Gretscher, René R.; Groth, Marco; Boland, Wilhelm; Burse, Antje

    2014-01-01

    Background Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC) carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi. Results In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp). RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration. Conclusion We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant

  9. A novel PET imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier

    PubMed Central

    Bankstahl, Jens P.; Bankstahl, Marion; Kuntner, Claudia; Stanek, Johann; Wanek, Thomas; Meier, Martin; Ding, Xiao-Qi; Müller, Markus; Langer, Oliver; Löscher, Wolfgang

    2013-01-01

    About one third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood-brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug-resistance in an individual patient. Using the radiolabeled P-glycoprotein substrate (R)-[11C]verapamil and different doses of co-administered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography (PET) can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood-brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 Tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[11C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k2, but not in distribution volume VT or influx rate constant K1, correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry. This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo. PMID:21677164

  10. Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

    PubMed

    Llaudó, Inés; Colom, Helena; Giménez-Bonafé, Pepita; Torras, Joan; Caldés, Anna; Sarrias, Maria; Cruzado, Josep M; Oppenheimer, Federico; Sánchez-Plumed, Jaime; Gentil, Miguel Ángel; Ekberg, Henrik; Grinyó, Josep M; Lloberas, Núria

    2013-02-01

    The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

  11. Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders.

    PubMed

    Qosa, Hisham; Miller, David S; Pasinelli, Piera; Trotti, Davide

    2015-12-01

    The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. This article is part of a Special Issue entitled SI: Neuroprotection.

  12. An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

    PubMed Central

    Leighton, J; Schatz, G

    1995-01-01

    In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth. Images PMID:7828591

  13. Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti.

    PubMed

    Nakano, Shigeru; Fukaya, Masahiro; Horinouchi, Sueharu

    2006-01-01

    Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH2-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.

  14. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

    PubMed Central

    Sasser, Terry L.; Padolina, Mark; Fratti, Rutilio A.

    2013-01-01

    Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. PMID:22970809

  15. P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

    PubMed

    Kooij, Gijs; Kroon, Jeffrey; Paul, Debayon; Reijerkerk, Arie; Geerts, Dirk; van der Pol, Susanne M A; van Het Hof, Bert; Drexhage, Joost A; van Vliet, Sandra J; Hekking, Liesbeth H P; van Buul, Jaap D; Pachter, Joel S; de Vries, Helga E

    2014-05-01

    The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

  16. Role of P-glycoprotein in refractoriness of seizures to antiepileptic drugs in Lennox-Gastaut syndrome.

    PubMed

    Kumar, Achal; Tripathi, Deepak; Paliwal, Vimal Kumar; Neyaz, Zafar; Agarwal, Vikas

    2015-02-01

    Mechanism of seizure refractoriness to antiepileptic drugs in children with Lennox-Gastaut syndrome is not known. Efflux of antiepileptic drugs due to increased expression/function of P-glycoprotein, a multidrug efflux transporter protein on the cell surface is a proposed mechanism. The authors studied the expression/function of P-glycoprotein on peripheral blood mononuclear cells of 29 children with Lennox-Gastaut syndrome, 23 children with other epilepsies, and 19 healthy children. The authors found a higher P-glycoprotein expression/function in Lennox-Gastaut syndrome, a higher percent positive cells as compared to children with other epilepsy (P < 0.001) and to healthy controls (P = 0.012), higher P-glycoprotein expression as compared to healthy controls (P = 0.003), a higher total P-glycoprotein expression (relative florescence intensity × percent positive cells) as compared to children with other epilepsies (P < 0.001) and healthy controls (P < 0.001), and a higher P-glycoprotein function as compared to children with other epilepsies (P = 0.001) and healthy controls (P = 0.002). These findings may explain seizure refractoriness to anti-epileptic drugs in Lennox-Gastaut syndome.

  17. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    SciTech Connect

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong; Li, Yunman; Huang, Wenlong

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  18. An overview of ABC and SLC drug transporter gene regulation.

    PubMed

    Chen, Qiu-Xia; Hu, Hai-Hong; Zhou, Quan; Yu, Ai-Ming; Zeng, Su

    2013-02-01

    Membrane transporters play a significant role in drug absorption, distribution and excretion, and they consequently affect the pharmacokinetics, efficacy and safety of a drug. Under certain circumstances, such as pathological processes or exposure to certain substances, the expression of drug transporters is modified in cells. Change in transporter expression and function may affect cellular drug disposition resulting in different drug responses. This raises a number of questions such as which drugs are likely to modulate the expression of drug transporters, what factors support this process, and which transporters are influenced in a particular situation. In this paper, we summarize recent findings to find an answer to these questions. Particularly, we present an overview of the transcription factors involved in the regulation of a given drug transporter, the signaling transduction pathways that contribute to drug transporter gene expression, and xenobiotics and endobiotics that initiate the processes.

  19. Three- and four-class classification models for P-glycoprotein inhibitors using counter-propagation neural networks.

    PubMed

    Thai, K-M; Huynh, N-T; Ngo, T-D; Mai, T-T; Nguyen, T-H; Tran, T-D

    2015-01-01

    P-glycoprotein (P-gp) is an ATP binding cassette (ABC) transporter that helps to protect several certain human organs from xenobiotic exposure. This efflux pump is also responsible for multi-drug resistance (MDR), an issue of the chemotherapy approach in the fight against cancer. Therefore, the discovery of P-gp inhibitors is considered one of the most popular strategies to reverse MDR in tumour cells and to improve therapeutic efficacy of commonly used cytotoxic drugs. Until now, several generations of P-gp inhibitors have been developed but they have largely failed in preclinical and clinical studies due to lack of selectivity, poor solubility and severe pharmacokinetic interactions. In this study, three models (SION, SIO, SIN) to classify specific 'true' P-gp inhibitors as well as three other models (CPBN, CPB1, CPN) to distinguish between P-gp inhibitors, CYP 3A inhibitors and co-inhibitors of these proteins with rather high accuracy values for the test set and the external set were generated based on counter-propagation neural networks (CPG-NN). Such three and four-class classification models helped provide more information about the bioactivities of compounds not only on one target (P-gp), but also on a combination of multiple targets (P-gp, CYP 3A).

  20. Structural basis for substrate specificity of an amino acid ABC transporter.

    PubMed

    Yu, Jie; Ge, Jingpeng; Heuveling, Johanna; Schneider, Erwin; Yang, Maojun

    2015-04-21

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

  1. Estimation of Candida albicans ABC Transporter Behavior in Real-Time via Fluorescence.

    PubMed

    Szczepaniak, Joanna; Łukaszewicz, Marcin; Krasowska, Anna

    2015-01-01

    We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

  2. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    NASA Astrophysics Data System (ADS)

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  3. Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ.

    PubMed

    Gouridis, Giorgos; Schuurman-Wolters, Gea K; Ploetz, Evelyn; Husada, Florence; Vietrov, Ruslan; de Boer, Marijn; Cordes, Thorben; Poolman, Bert

    2015-01-01

    The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

  4. Segmental dependent transport of low permeability compounds along the small intestine due to P-glycoprotein: the role of efflux transport in the oral absorption of BCS class III drugs.

    PubMed

    Dahan, Arik; Amidon, Gordon L

    2009-01-01

    The purpose of this study was to investigate the role of P-gp efflux in the in vivo intestinal absorption process of BCS class III P-gp substrates, i.e. high-solubility low-permeability drugs. The in vivo permeability of two H (2)-antagonists, cimetidine and famotidine, was determined by the single-pass intestinal perfusion model in different regions of the rat small intestine, in the presence or absence of the P-gp inhibitor verapamil. The apical to basolateral (AP-BL) and the BL-AP transport of the compounds in the presence or absence of various efflux transporters inhibitors (verapamil, erythromycin, quinidine, MK-571 and fumitremorgin C) was investigated across Caco-2 cell monolayers. P-gp expression levels in the different intestinal segments were confirmed by immunoblotting. Cimetidine and famotidine exhibited segmental dependent permeability through the gut wall, with decreased P(eff) in the distal ileum in comparison to the proximal regions of the intestine. Coperfusion of verapamil with the drugs significantly increased the permeability in the ileum, while no significant change in the jejunal permeability was observed. Both drugs exhibited significantly greater BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Concentration dependent decrease of this secretion was obtained by the P-gp inhibitors verapamil, erythromycin and quinidine, while no effect was evident by the MRP2 inhibitor MK-571 and the BCRP inhibitor FTC, indicating that P-gp is the transporter mediates the intestinal efflux of cimetidine and famotidine. P-gp levels throughout the intestine were inversely related to the in vivo permeability of the drugs from the different segments. The data demonstrate that for these high-solubility low-permeability P-gp substrates, P-gp limits in vivo intestinal absorption in the distal segments of the small intestine; however P-gp plays a minimal role in the proximal intestinal segments due to significant lower P-gp expression levels

  5. ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria.

    PubMed

    Narita, Shin-ichiro

    2011-01-01

    The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed. PMID:21670534

  6. Correction: Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2016-04-15

    The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABCB subfamily display very high substrate specificity compared with their mammalian counterparts that are often associated with multidrug resistance (MDR) phenomena. In this review we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plantArabidopsisreveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins (FKBPs) that chaperon both transporters to the plasma membrane in an action that seems to involve Hsp90. Further both transporters are phosphorylated at regulatory domains that connect both nucleotide-binding folds. Taken together it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI). PMID:27068986

  7. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  8. A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

    PubMed

    Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

    2009-02-01

    Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall. PMID:19244140

  9. Clinico-Pathologic Function of Cerebral ABC Transporters – Implications for the Pathogenesis of Alzheimer’s Disease

    PubMed Central

    Pahnke, Jens; Wolkenhauer, Olaf; Krohn, Markus; Walker, Lary C.

    2009-01-01

    In recent years it has become evident that ABC transporters fulfill important barrier functions in normal organs and during disease processes. Most importantly, resistance to drugs in cancer cells led to intense oncological and pharmacological investigations in which researchers were able to highlight important pharmacological interactions of chemotherapeuticals with ABC transporter function. Recently, the development of neurodegenerative diseases and the maintenance of neuronal stem cells have been linked to the activity of ABC transporters. Here, we summarize findings from cell culture experiments, animal models and studies of patients with Alzheimer’s disease. Furthermore, we discuss pharmacological interactions and computational methods for risk assessment. PMID:18690837

  10. Synthesis and P-glycoprotein induction activity of colupulone analogs.

    PubMed

    Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

    2015-05-21

    Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent.

  11. Synthesis and P-glycoprotein induction activity of colupulone analogs.

    PubMed

    Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

    2015-05-21

    Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent. PMID:25875530

  12. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone.

    PubMed

    Kao, Yu-Han; Chern, Yijuang; Yang, Hui-Ting; Chen, Hui-Mei; Lin, Chun-Jung

    2016-08-01

    Huntington's disease (HD) is a neurodegenerative disease marked by an expanded polyglutamine (polyQ) tract on the huntingtin (HTT) protein that may cause transcriptional dysfunction. This study aimed to investigate the regulation and function of P-glycoprotein, an important efflux transporter, in brain capillaries in HD. The results showed that, compared with the littermate controls, R6/2 HD transgenic mice with the human mutant HTT gene had higher levels of P-glycoprotein mRNA and protein and enhanced NF-κB activity in their brain capillaries. Higher P-glycoprotein expression was also observed in the brain capillaries of human HD patients. Consistent with this enhanced P-glycoprotein expression, brain extracellular levels and brain-to-plasma ratios of the antipsychotic agents risperidone and paliperidone were significantly lower in R6/2 mice than in their littermate controls. Exogenous expression of human mutant HTT protein with expanded polyQ (mHTT-109Q) in HEK293T cells enhanced the levels of P-glycoprotein transcripts and NF-κB activity compared with cells expressing normal HTT-25Q. Treatment with the IKK inhibitor, BMS-345541, decreased P-glycoprotein mRNA level in cells transfected with mHTT-109Q or normal HTT-25Q In conclusion, mutant HTT altered the expression of P-glycoprotein through the NF-κB pathway in brain capillaries in HD and markedly affected the availability of P-glycoprotein substrates in the brain.

  13. Nucleotide-induced conformational dynamics in ABC transporters from structure-based coarse grained modelling.

    NASA Astrophysics Data System (ADS)

    Flechsig, Holger

    2016-02-01

    ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter

  14. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

    NASA Astrophysics Data System (ADS)

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-12-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

  15. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi.

    PubMed

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-01-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps. PMID:25504146

  16. A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase

    PubMed Central

    Morisaki, J. Hiroshi; Smith, Peter A.; Date, Shailesh V.; Kajihara, Kimberly K.; Truong, Chau Linda; Modrusan, Zora; Yan, Donghong; Kang, Jing; Xu, Min; Shah, Ishita M.; Mintzer, Robert; Kofoed, Eric M.; Cheung, Tommy K.; Arnott, David; Koehler, Michael F. T.; Heise, Christopher E.; Brown, Eric J.

    2016-01-01

    ABSTRACT The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro. These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB. Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo. PMID:27601569

  17. Substrate binding by a bacterial ABC transporter involved in polysaccharide export

    SciTech Connect

    Cuthbertson, Leslie; Kimber, Matthew S.; Whitfield, Chris

    2008-04-02

    ATP-binding-cassette (ABC) transporters are responsible for the export of a wide variety of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria. These include the O-antigenic polysaccharide (O-PS) portion of lipopolysaccharide, a crucial virulence determinant in Gram-negative pathogens. O-PSs are synthesized by one of two fundamentally different pathways. Escherichia coli O serotypes O8 and O9a provide the prototype systems for studying O-PS export via ABC transporters. The transporter is composed of the transmembrane component Wzm and the nucleotide-binding component Wzt. Although the N-terminal domain of Wzt is a conventional ABC protein, the C-terminal domain of Wzt (C-Wzt) is a unique structural element that determines the specificity of the transporter for either the O8 or O9a O-PS. We show here that the two domains of Wzt can function when expressed as separate polypeptides; both are essential for export. In vitro, C-Wzt binds its cognate O-PS by recognizing a residue located at the nonreducing end of the polymer. The crystal structure of C-WztO9a is reported here and reveals a {beta} sandwich with an immunoglobulin-like topology that contains the O-PS-binding pocket. Substrate interactions with nucleotide-binding domains have been demonstrated in an ABC exporter previously. However, to our knowledge substrate binding by a discrete, cytoplasmic accessory domain in an extended nucleotide-binding domain polypeptide has not previously been demonstrated. Elucidation of the substrate-recognition system involved in O-PS export provides insight into the mechanism that coordinates polymer biosynthesis, termination, and export.

  18. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed Central

    Theodoulou, Frederica L.; Carrier, David J.; Schaedler, Theresia A.; Baldwin, Stephen A.; Baker, Alison

    2016-01-01

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  19. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed

    Theodoulou, Frederica L; Carrier, David J; Schaedler, Theresia A; Baldwin, Stephen A; Baker, Alison

    2016-06-15

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  20. Role of the Zinc Uptake ABC Transporter of Moraxella catarrhalis in Persistence in the Respiratory Tract

    PubMed Central

    Brauer, Aimee L.; Kirkham, Charmaine; Johnson, Antoinette; Koszelak-Rosenblum, Mary; Malkowski, Michael G.

    2013-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains of M. catarrhalis tested. A mutant in which the znu gene cluster is knocked out shows markedly impaired growth compared to the wild type in medium that contains trace zinc; growth is restored to wild-type levels by supplementing medium with zinc but not with other divalent cations. Thermal-shift assays showed that the purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter of M. catarrhalis is critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir of M. catarrhalis is present in the human respiratory tract and this reservoir is important for persistence. The znu knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments with very low zinc concentrations and is critical for full virulence of M. catarrhalis in the respiratory tract in facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract. PMID:23817618

  1. Alzheimer’s and ABC transporters - new opportunities for diagnostics and treatment

    PubMed Central

    Pahnke, Jens; Langer, Oliver; Krohn, Markus

    2014-01-01

    Much has been said about the increasing number of demented patients and the main risk factor ‘age’. Frustratingly, we do not know the precise pattern and all modulating factors that provoke the pathologic changes in the brains of affected elderly. We have to diagnose early to be able to stop the progression of diseases that irreversibly destroy brain substance. Familiar AD cases have mislead some researchers for almost 20 years, which has unfortunately narrowed the scientific understanding and has, thus, lead to insufficient funding of independent approaches. Therefore, basic researchers hardly have been able to develop causative treatments and clinicians still do not have access to prognostic and early diagnostic tools. During the recent years it became clear that insufficient Aβ export, physiologically facilitated by the ABC transporter superfamily at the brain’s barriers, plays a fundamental role in disease initiation and progression. Furthermore, export mechanisms that are deficient in affected elderly are new targets for activation and, thus, treatment, but ideally also for prevention. In sporadic AD disturbed clearance of β-amyloid from the brain is so far the most important factor for its accumulation in the parenchyma and vessel walls. Here, we review findings about the contribution of ABC transporters and of the perivascular drainage/glymphatic system on β-amyloid clearance. We highlight their potential value for innovative early diagnostics using PET and describe recently described, effective ABC transporter-targeting agents as potential causative treatment for neurodegenerative proteopathies/dementias. PMID:24746857

  2. Release of Entropic Spring Reveals Conformational Coupling Mechanism in the ABC Transporter BtuCD-F.

    PubMed

    Prieß, Marten; Schäfer, Lars V

    2016-06-01

    Substrate translocation by ATP-binding cassette (ABC) transporters involves coupling of ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) to conformational changes in the transmembrane domains. We used molecular dynamics simulations to investigate the atomic-level mechanism of conformational coupling in the ABC transporter BtuCD-F, which imports vitamin B12 across the inner membrane of Escherichia coli. Our simulations show how an engineered disulfide bond across the NBD dimer interface reduces conformational fluctuations and hence configurational entropy. As a result, the disulfide bond is under substantial mechanical stress. Releasing this entropic spring, as is the case in the wild-type transporter, combined with analyzing the pairwise forces between individual residues, unravels the coupling mechanism. The identified pathways along which force is propagated from the NBDs via the coupling helix to the transmembrane domains are composed of highly conserved residues, underlining their functional relevance. This study not only reveals the details of conformational coupling in BtuCD-F, it also provides a promising approach to other long-range conformational couplings, e.g., in ABC exporters or other ATP-driven molecular machines.

  3. Release of Entropic Spring Reveals Conformational Coupling Mechanism in the ABC Transporter BtuCD-F.

    PubMed

    Prieß, Marten; Schäfer, Lars V

    2016-06-01

    Substrate translocation by ATP-binding cassette (ABC) transporters involves coupling of ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) to conformational changes in the transmembrane domains. We used molecular dynamics simulations to investigate the atomic-level mechanism of conformational coupling in the ABC transporter BtuCD-F, which imports vitamin B12 across the inner membrane of Escherichia coli. Our simulations show how an engineered disulfide bond across the NBD dimer interface reduces conformational fluctuations and hence configurational entropy. As a result, the disulfide bond is under substantial mechanical stress. Releasing this entropic spring, as is the case in the wild-type transporter, combined with analyzing the pairwise forces between individual residues, unravels the coupling mechanism. The identified pathways along which force is propagated from the NBDs via the coupling helix to the transmembrane domains are composed of highly conserved residues, underlining their functional relevance. This study not only reveals the details of conformational coupling in BtuCD-F, it also provides a promising approach to other long-range conformational couplings, e.g., in ABC exporters or other ATP-driven molecular machines. PMID:27276259

  4. Role of ATP-binding cassette (ABC) transporters in interactions between natural products and drugs.

    PubMed

    Aszalos, Adorjan

    2008-12-01

    Medicinal use of natural products such as extracts of plants has existed for many years in China and in other countries and they are now available worldwide. Citrus fruit juices are consumed on a daily basis around the world. Modern medicine provides well-tested compounds or drugs for most sicknesses. However, the simultaneous consumption of plant extracts, food supplements, and fruit juices with drugs can create metabolic aberrations in humans. Interactions between drugs used simultaneously are regulated by government agencies. Not regulated, but warned against in drug inserts are potential interactions between drugs and food and food-additives containing certain compounds with potential side effects. Summarized here are the results of investigations that point out possible interactions at the level of transporter molecules by drugs and compounds of natural origin. These transporter molecules play important roles in absorption in the intestines, at the blood brain barrier, in the liver, the kidney and in some other parts of the human body. Drugs and metabolites pass through these pumps and may compete with compounds from food supplements. The most studied natural compounds that are potential modulators of these transport molecules are flavonoids, found in fruit juices, vegetables, flowers and tea. Mycotoxins found in cereal grains are also shown to modulate transporter proteins. We detail here how such constituents of natural origin were shown to modulate three types of the major transporter molecules, P-glycoprotein (ABCB1), multidrug resistance proteins (ABCCs) and breast cancer resistance protein (ABCG2). Interference of these natural compounds with drugs at the transporter level is also discussed. PMID:19075617

  5. Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus.

    PubMed

    Blackhall, W J; Liu, H Y; Xu, M; Prichard, R K; Beech, R N

    1998-09-15

    Resistance to anthelmintics that are used to control parasite populations in domestic animals has become a serious problem worldwide. The development of resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. The anthelmintic ivermectin is known to bind to the human membrane transport protein, P-glycoprotein, and P-glycoprotein-deficient mice treated with ivermectin have shown signs of neurotoxicity. P-glycoprotein is believed to be involved in the multidrug resistance phenotype seen in some human cancers and for drug resistance in some protists. We have examined the genetic variation of a P-glycoprotein homologue from the nematode Haemonchus contortus to see if an association exists between specific alleles of this gene and survival to exposure to ivermectin or moxidectin. Two parasite strains passaged without drug treatment and three strains, subjected to anthelmintic selection and derived from the unselected strains, were examined. Allelic variation in the unselected strains showed this locus to be highly polymorphic. chi 2 analyses of allele frequencies showed significant differences between the unselected and the drug-selected derived strains. In all three drug-selected strains, an apparent selection for the same allele was observed. These findings suggest that P-glycoprotein may be involved in resistance to both ivermectin and moxidectin in H. contortus.

  6. Molecular insight into conformational transmission of human P-glycoprotein

    SciTech Connect

    Chang, Shan-Yan; Liu, Fu-Feng E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan E-mail: ysun@tju.edu.cn

    2013-12-14

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  7. Molecular insight into conformational transmission of human P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Chang, Shan-Yan; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

    2013-12-01

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  8. Active transporters as enzymes: an energetic framework applied to major facilitator superfamily and ABC importer systems.

    PubMed

    Shilton, Brian H

    2015-04-15

    Active membrane transporters are dynamic molecular machines that catalyse transport across a membrane by coupling solute movement to a source of energy such as ATP or a secondary ion gradient. A central question for many active transporters concerns the mechanism by which transport is coupled to a source of energy. The transport process and associated energetic coupling involve conformational changes in the transporter. For efficient transport, the conformational changes must be tightly regulated and they must link energy use to movement of the substrate across the membrane. The present review discusses active transport using the well-established energetic framework for enzyme-mediated catalysis. In particular, membrane transport systems can be viewed as ensembles consisting of low-energy and high-energy conformations. The transport process involves binding interactions that selectively stabilize the higher energy conformations, and in this way promote conformational changes in the system that are coupled to decreases in free energy and substrate translocation. The major facilitator superfamily of secondary active transporters is used to illustrate these ideas, which are then be expanded to primary active transport mediated by ABC (ATP-binding cassette) import systems, with a focus on the well-studied maltose transporter.

  9. Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

    PubMed

    Mackenzie, S M; Brooker, M R; Gill, T R; Cox, G B; Howells, A J; Ewart, G D

    1999-07-15

    The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters. PMID:10407069

  10. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture.

    PubMed

    Follit, Courtney A; Brewer, Frances K; Wise, John G; Vogel, Pia D

    2015-10-01

    Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716-726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances. PMID:26516582

  11. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture

    PubMed Central

    Follit, Courtney A; Brewer, Frances K; Wise, John G; Vogel, Pia D

    2015-01-01

    Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716–726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances. PMID:26516582

  12. Michaelis-Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein.

    PubMed

    Meyer, Markus R; Orschiedt, Tina; Maurer, Hans H

    2013-02-27

    The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug-drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug-drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis-Menten kinetics and assessing their K(m) and V(max) values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with K(m) and V(max) values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged. PMID:23273999

  13. Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

    PubMed

    Ahuja, Shivani; Rougé, Lionel; Swem, Danielle L; Sudhamsu, Jawahar; Wu, Ping; Russell, Stephen J; Alexander, Mary Kate; Tam, Christine; Nishiyama, Mireille; Starovasnik, Melissa A; Koth, Christopher M

    2015-04-01

    Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.

  14. ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence

    PubMed Central

    Murphy, Timothy F; Brauer, Aimee L.; Johnson, Antoinette; Kirkham, Charmaine

    2016-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. PMID:27391026

  15. ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence.

    PubMed

    Murphy, Timothy F; Brauer, Aimee L; Johnson, Antoinette; Kirkham, Charmaine

    2016-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. PMID:27391026

  16. Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus.

    PubMed

    Song, Ting-Ting; Zhao, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    Multidrug resistance (MDR) confers agrochemical compatibility to fungal cells-based mycoinsecticdes but mechanisms involved in MDR remain poorly understood for entomopathogenic fungi, which have been widely applied as biocontrol agents against arthropod pests. Here we characterized the functions of five ATP-binding cassette (ABC) transporters, which were classified to the subfamilies ABC-B (Mdr1), ABC-C (Mrp1) and ABC-G (Pdr1, Pdr2 and Pdr5) and selected from 54 full-size ABC proteins of Beauveria bassiana based on their main domain architecture, membrane topology and transcriptional responses to three antifungal inducers. Disruption of each transporter gene resulted in significant reduction in resistance to four to six of eight fungicides or antifungal drugs tested due to their differences in structure and function. Compared with wild-type and complemented (control) strains, disruption mutants of all the five transporter genes became significantly less tolerant to the oxidants menadione and H₂O₂ based on 22-41% and 10-31% reductions of their effective concentrations required for the suppression of 50% colony growth at 25°C. Under a standardized spray, the killing actions of ΔPdr5 and ΔMrp1 mutants against Spodoptera litura second-instar larvae were delayed by 59% and 33% respectively. However, no significant virulence change was observed in three other delta mutants. Taken together, the examined five ABC transporters contribute differentially to not only the fungal MDR but antioxidant capability, a phenotype rarely associated with ABC efflux pumps in previous reports; at least some of them are required for the full virulence of B. bassiana, thereby affecting the fungal biocontrol potential. Our results indicate that ABC pump-dependent MDR mechanisms exist in entomopathogenic fungi as do in yeasts and human and plant pathogenic fungi.

  17. Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design

    NASA Astrophysics Data System (ADS)

    Ishikawa, Toshihisa; Tamura, Ai; Saito, Hikaru; Wakabayashi, Kanako; Nakagawa, Hiroshi

    2005-10-01

    In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

  18. Evidence that Bacterial ABC-Type Transporter Imports Free EDTA for Metabolism

    SciTech Connect

    Zhang, Hua; Herman, Jacob P.; Bolton, Harvey; Zhang, Zhicheng; Clark, Sue B.; Xun, Luying

    2007-11-01

    Ethylenediaminetetraacetic acid (EDTA), a common chelating agent, is becoming a major organic pollutant in the form of metal-EDTA complexes in surface waters, partly due to its recalcitrance to biodegradation. Even an EDTA-degrading bacterium BNC1 does not degrade stable metal-EDTA complexes. An ABC-type transporter was identified for possible uptake of EDTA because the transporter genes and EDTA monooxygenase gene were expressed in a single operon in BNC1. The ABC-type transporter had a periplasmic binding protein (EppA) that should confer the substrate specificity for the transporter; therefore, EppA was produced in Escherichia coli,purified, and characterized. EppA was shown to bind free EDTA with a dissociation constant as low as 25 nM by using isothermal titration calorimetry. When unstable metal-EDTA complexes, e.g. MgEDTA2-, were added to the EppA solution, binding was also observed. However, experimental data and theoretical analysis only supported EppA binding of free EDTA. When stable metal-EDTA complexes, e.g. CuEDTA2-, are titrated into the EppA solution, no binding was observed. Since EDTA monooxygenase in the cytoplasm uses some of the stable metal-EDTA complexes as substrates, we suggest that the lack of EppA binding and EDTA uptake are responsible for the failure of BNC1 cells to degrade the stable complexes.

  19. An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

    Gahan, Linda J.; Pauchet, Yannick; Vogel, Heiko; Heckel, David G.

    2010-01-01

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field. PMID:21187898

  20. P-glycoprotein Inhibition by the Agricultural Pesticide Propiconazole and Its Hydroxylated Metabolites: Implications for Pesticide-Drug Interactions.

    EPA Science Inventory

    The human efflux transporter P-glycoprotein (P-gp; MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure ass...

  1. P-glycoprotein Inhibition by the Agricultural Pesticide Propiconazole and Its Hydroxylated Metabolites: Implications for Pesticide-Drug Interactions

    EPA Science Inventory

    The human efflux transporter P-glycoprotein (P-gp, MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure ass...

  2. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    PubMed Central

    Hürlimann, Lea M.; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V.; Tieleman, D. Peter

    2016-01-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  3. A Silent ABC Transporter Isolated from Streptomyces rochei F20 Induces Multidrug Resistance

    PubMed Central

    Fernández-Moreno, Miguel A.; Carbó, Lázaro; Cuesta, Trinidad; Vallín, Carlos; Malpartida, Francisco

    1998-01-01

    In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3′ end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3′ terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter. PMID:9696745

  4. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    PubMed Central

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-01-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate. PMID:25377891

  5. Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana.

    PubMed

    Kolukisaoglu, H Uner; Bovet, Lucien; Klein, Markus; Eggmann, Thomas; Geisler, Markus; Wanke, Dierk; Martinoia, Enrico; Schulz, Burkhard

    2002-11-01

    Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers. PMID:12430019

  6. Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation.

    PubMed

    Mendiondo, Guillermina M; Medhurst, Anne; van Roermund, Carlo W; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R; Waugh, Robbie; Theodoulou, Frederica L; Holdsworth, Michael J

    2014-09-01

    In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ.

  7. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    SciTech Connect

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  8. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

  9. ABC-Transporter Expression Does Not Correlate with Response to Irinotecan in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Trumpi, K.; Emmink, B.L.; Prins, A.M.; van Oijen, M.G.H.; van Diest, P.J.; Punt, C.J.A.; Koopman, M.; Kranenburg, O.; Rinkes, I.H.M. Borel

    2015-01-01

    Background: Active efflux of irinotecan by ATP-binding cassette (ABC)-transporters, in particular ABCB1 and ABCG2, is a well-established drug resistance mechanism in vitro and in pre-clinical mouse models, but its relevance in colorectal cancer (CRC) patients is unknown. Therefore, we assessed the association between ABC-transporter expression and tumour response to irinotecan in patients with metastatic CRC. Methods: Tissue microarrays of a large cohort of metastatic CRC patients treated with irinotecan in a prospective study (CAIRO study; n=566) were analysed for expression of ABCB1 and ABCG2 by immunohistochemistry. Kaplan-Meier and Cox proportional hazard regression analyses were performed to assess the association of ABC transporter expression with irinotecan response. Gene expression profiles of 17 paired tumours were used to assess the concordance of ABCB1/ABCG2 expression in primary CRC and corresponding metastases. Results: The response to irinotecan was not significantly different between primary tumours with positive versus negative expression of ABCB1 (5.8 vs 5.7 months, p=0.696) or ABCG2 (5.7 vs 6.1 months, p=0.811). Multivariate analysis showed neither ABCB1 nor ABCG2 were independent predictors for progression free survival. There was a mediocre to poor concordance between ABC-transporter expression in paired tumours. Conclusion: In metastatic CRC, ABC-transporter expression in the primary tumour does not predict irinotecan response. PMID:26516354

  10. Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path–Force Matching QM/MM Method

    PubMed Central

    Zhou, Y.; Ojeda-May, P.; Nagaraju, M.; Pu, J.

    2016-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are ubiquitous ATP-dependent membrane proteins involved in translocations of a wide variety of substrates across cellular membranes. To understand the chemomechanical coupling mechanism as well as functional asymmetry in these systems, a quantitative description of how ABC transporters hydrolyze ATP is needed. Complementary to experimental approaches, computer simulations based on combined quantum mechanical and molecular mechanical (QM/MM) potentials have provided new insights into the catalytic mechanism in ABC transporters. Quantitatively reliable determination of the free energy requirement for enzymatic ATP hydrolysis, however, requires substantial statistical sampling on QM/MM potential. A case study shows that brute force sampling of ab initio QM/MM (AI/MM) potential energy surfaces is computationally impractical for enzyme simulations of ABC transporters. On the other hand, existing semiempirical QM/MM (SE/MM) methods, although affordable for free energy sampling, are unreliable for studying ATP hydrolysis. To close this gap, a multiscale QM/MM approach named reaction path–force matching (RP–FM) has been developed. In RP–FM, specific reaction parameters for a selected SE method are optimized against AI reference data along reaction paths by employing the force matching technique. The feasibility of the method is demonstrated for a proton transfer reaction in the gas phase and in solution. The RP–FM method may offer a general tool for simulating complex enzyme systems such as ABC transporters. PMID:27498639

  11. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae

    PubMed Central

    Lee, Youngjin

    2016-01-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn’t show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  12. Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path-Force Matching QM/MM Method.

    PubMed

    Zhou, Y; Ojeda-May, P; Nagaraju, M; Pu, J

    2016-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are ubiquitous ATP-dependent membrane proteins involved in translocations of a wide variety of substrates across cellular membranes. To understand the chemomechanical coupling mechanism as well as functional asymmetry in these systems, a quantitative description of how ABC transporters hydrolyze ATP is needed. Complementary to experimental approaches, computer simulations based on combined quantum mechanical and molecular mechanical (QM/MM) potentials have provided new insights into the catalytic mechanism in ABC transporters. Quantitatively reliable determination of the free energy requirement for enzymatic ATP hydrolysis, however, requires substantial statistical sampling on QM/MM potential. A case study shows that brute force sampling of ab initio QM/MM (AI/MM) potential energy surfaces is computationally impractical for enzyme simulations of ABC transporters. On the other hand, existing semiempirical QM/MM (SE/MM) methods, although affordable for free energy sampling, are unreliable for studying ATP hydrolysis. To close this gap, a multiscale QM/MM approach named reaction path-force matching (RP-FM) has been developed. In RP-FM, specific reaction parameters for a selected SE method are optimized against AI reference data along reaction paths by employing the force matching technique. The feasibility of the method is demonstrated for a proton transfer reaction in the gas phase and in solution. The RP-FM method may offer a general tool for simulating complex enzyme systems such as ABC transporters. PMID:27498639

  13. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae.

    PubMed

    Lee, Youngjin

    2016-02-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn't show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  14. P-glycoprotein activity in the blood-brain barrier is affected by virus-induced neuroinflammation and antipsychotic treatment.

    PubMed

    Doorduin, Janine; de Vries, Erik F J; Dierckx, Rudi A; Klein, Hans C

    2014-10-01

    A large percentage of schizophrenic patients respond poorly to antipsychotic treatment. This could be explained by inefficient drug transport across the blood-brain barrier due to P-glycoprotein mediated efflux. P-glycoprotein activity and expression in the blood-brain barrier can be affected by inflammation and pharmacotherapy. We therefore investigated the effect of herpes simplex virus type-1 (HSV-1) induced neuroinflammation and antipsychotic treatment on P-glycoprotein activity. Rats were inoculated with HSV-1 or PBS (control) on day 0 and treated with saline, clozapine or risperidone from day 0 up until day 4 post-inoculation. Positron emission tomography with the P-glycoprotein substrate [11C]verapamil was used to assess P-glycoprotein activity at day 6 post-inoculation. Disease symptoms in HSV-1 inoculated rats increased over time and were not significantly affected by treatment. The volume of distribution (VT) of [11C]verapamil was significantly lower (10-22%) in HSV-1 inoculated rats than in control rats. In addition, antipsychotic treatment significantly affected the VT of [11C]verapamil in all brain regions, although this effect was drug dependent. In fact, VT of [11C]verapamil was significantly increased (22-39%) in risperidone treated rats in most brain regions when compared to clozapine treated rats and in midbrain when compared to saline treated rats. No interaction between HSV-1 inoculation and antipsychotic treatment on VT of [11C]verapamil was found. In this study we demonstrated that HSV-1 induced neuroinflammation increased and risperidone treatment decreased P-glycoprotein activity. This finding is of importance for the understanding of treatment resistance in schizophrenia, and warrants further investigation of the underlying mechanism and the importance in clinical practice.

  15. Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli.

    PubMed

    Reimann, Sven; Poschmann, Gereon; Kanonenberg, Kerstin; Stühler, Kai; Smits, Sander H J; Schmitt, Lutz

    2016-08-15

    Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB. PMID:27279651

  16. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence

    PubMed Central

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M.; Singh, Ashutosh; Coste, Alix T.; Andes, David R.; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-01-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans. Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  17. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

    PubMed

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M; Singh, Ashutosh; Coste, Alix T; Andes, David R; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-06-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  18. ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

    PubMed Central

    Videau, Patrick; Rivers, Orion S.; Higa, Kelly C.

    2015-01-01

    ABSTRACT In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. IMPORTANCE How chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was

  19. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    PubMed

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  20. [Role of the ABC transporters A1 and G1, key reverse cholesterol transport proteins, in atherosclerosis].

    PubMed

    Demina, E P; Miroshnikova, V V; Schwarzman, A L

    2016-01-01

    Atherosclerosis is one of the most common causes of death worldwide. Epidemiology studies firmly established an inverse relationship between atherogenesis and distorted lipid metabolism, in particular, higher levels of total cholesterol, an accumulation of CH-laden macrophages (foam cells), and lower plasma levels of antiatherogenic high density lipoprotein (HDL). It is believed that the reverse cholesterol transport, a process that removes excess cholesterol from peripheral tissues/cells including macrophages to circulating HDL, is one of the main mechanisms responsible for anti-atherogenic properties of HDL. The key proteins of reverse cholesterol transport-ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1)-mediate the cholesterol efflux from macrophages and prevent their transformation into foam cells. This review focuses on the role of ABC transporters A1 and G1 in the pathogenesis of atherosclerosis.

  1. Overexpression, Membrane Preparation, and Purification of a Typical Multidrug ABC Transporter BmrA.

    PubMed

    Wiseman, Benjamin; Jault, Jean-Michel

    2016-01-01

    The production and purification is normally the first step in any biophysical or biochemical study of a new target protein. For membrane proteins, due to their generally low expression levels and hydrophobic properties this is often a major hurdle. Some multidrug transporters are members of one of the largest families of membrane proteins, the ABC ("ATP-binding cassette"), and are responsible for the uptake and export of a wide variety of molecules. This can lead to resistance when those molecules are antibiotics or chemotherapy drugs. To better understand their role in multidrug resistance pure and active protein is required. Here we outline a protocol to produce a highly pure and functionally active multidrug transporter BmrA that is suitable for use in biophysical and biochemical studies. We show that BmrA can be heterologously overexpressed in huge amount in E. coli and extracted from the membrane in a functionally active form. PMID:27485334

  2. The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm[W

    PubMed Central

    Landgraf, Ramona; Smolka, Ulrike; Altmann, Simone; Eschen-Lippold, Lennart; Senning, Melanie; Sonnewald, Sophia; Weigel, Benjamin; Frolova, Nadezhda; Strehmel, Nadine; Hause, Gerd; Scheel, Dierk; Böttcher, Christoph; Rosahl, Sabine

    2014-01-01

    The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components. PMID:25122151

  3. Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio).

    PubMed

    Liu, Xiang; Li, Shangqi; Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A; Xu, Peng

    2016-01-01

    The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.

  4. Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio).

    PubMed

    Liu, Xiang; Li, Shangqi; Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A; Xu, Peng

    2016-01-01

    The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp. PMID:27058731

  5. Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio)

    PubMed Central

    Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A.

    2016-01-01

    The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp. PMID:27058731

  6. Inhibition of ABC transport proteins by oil sands process affected water.

    PubMed

    Alharbi, Hattan A; Saunders, David M V; Al-Mousa, Ahmed; Alcorn, Jane; Pereira, Alberto S; Martin, Jonathan W; Giesy, John P; Wiseman, Steve B

    2016-01-01

    The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates

  7. Inhibition of ABC transport proteins by oil sands process affected water.

    PubMed

    Alharbi, Hattan A; Saunders, David M V; Al-Mousa, Ahmed; Alcorn, Jane; Pereira, Alberto S; Martin, Jonathan W; Giesy, John P; Wiseman, Steve B

    2016-01-01

    The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates

  8. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

    PubMed Central

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H.; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers. PMID:27028005

  9. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

    PubMed

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers. PMID:27028005

  10. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

    PubMed

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.

  11. Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation

    PubMed Central

    Mendiondo, Guillermina M.; Medhurst, Anne; van Roermund, Carlo W.; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R.; Waugh, Robbie; Theodoulou, Frederica L.; Holdsworth, Michael J.

    2014-01-01

    In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ. PMID:24913629

  12. Increased intestinal P-glycoprotein expression and activity with progression of diabetes and its modulation by epigallocatechin-3-gallate: Evidence from pharmacokinetic studies.

    PubMed

    Dash, Ranjeet Prasad; Ellendula, Bhanuchander; Agarwal, Milee; Nivsarkar, Manish

    2015-11-15

    The aim of this study was to evaluate the change in the expression and the activity of intestinal P-glycoprotein (efflux transporter) with progression of diabetes in rats. Diabetes was induced in Wistar rats using a combination of low dose streptozotocin along with high fat diet. The expression of intestinal P-glycoprotein significantly increased (P≤0.05) with the progression of diabetes which was inferred from the mRNA analysis of mdr1a and mdr1b genes in the ileum segment of rat intestine. Furthermore, a significant increase (P≤0.05) in Na(+)-K(+) ATPase activity was observed in the ileum segment of rat intestine with the progression of diabetes. As a result of this, a significant decrease in the intestinal uptake and peroral bioavailability of the P-glycoprotein substrates (verapamil and atorvastatin) was observed along with the progression of diabetes as compared to normal animals. To address this problem of impaired drug uptake and bioavailability, a reported P-glycoprotein inhibitor, epigallocatechin-3-gallate, was experimentally evaluated. The treatment with epigallocatechin-3-gallate resulted in significant reduction in the expression and activity of P-glycoprotein and subsequent improvement in the intestinal uptake and peroral bioavailability of both verapamil and atorvastatin in normal as well as in diabetic animals. The findings of this study rationalised the use and established the mechanism of action of epigallocatechin-3-gallate to overcome P-glycoprotein mediated drug efflux and will also be helpful in therapeutic drug monitoring in diabetes.

  13. St. John's Wort reduces beta-amyloid accumulation in a double transgenic Alzheimer's disease mouse model-role of P-glycoprotein.

    PubMed

    Brenn, Anja; Grube, Markus; Jedlitschky, Gabriele; Fischer, Andrea; Strohmeier, Barbara; Eiden, Martin; Keller, Markus; Groschup, Martin H; Vogelgesang, Silke

    2014-01-01

    The adenosine triphosphate-binding cassette transport protein P-glycoprotein (ABCB1) is involved in the export of beta-amyloid from the brain into the blood, and there is evidence that age-associated deficits in cerebral P-glycoprotein content may be involved in Alzheimer's disease pathogenesis. P-glycoprotein function and expression can be pharmacologically induced by a variety of compounds including extracts of Hypericum perforatum (St. John's Wort). To clarify the effect of St. John's Wort on the accumulation of beta-amyloid and P-glycoprotein expression in the brain, St. John's Wort extract (final hyperforin concentration 5%) was fed to 30-day-old male C57BL/6J-APP/PS1(+/-) mice over a period of 60 or 120 days, respectively. Age-matched male C57BL/6J-APP/PS1(+/-) mice receiving a St. John's Wort-free diet served as controls. Mice receiving St. John's Wort extract showed (i) significant reductions of parenchymal beta-amyloid 1-40 and 1-42 accumulation; and (ii) moderate, but statistically significant increases in cerebrovascular P-glycoprotein expression. Thus, the induction of cerebrovascular P-glycoprotein may be a novel therapeutic strategy to protect the brain from beta-amyloid accumulation, and thereby impede the progression of Alzheimer's disease.

  14. PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid

    PubMed Central

    Kang, Joohyun; Hwang, Jae-Ung; Kim, Yu-Young; Assmann, Sarah M.; Martinoia, Enrico; Lee, Youngsook

    2010-01-01

    Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants. PMID:20133880

  15. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain.

    PubMed

    Soares, Ricardo V; Do, Tuan M; Mabondzo, Aloïse; Pons, Gérard; Chhun, Stéphanie

    2016-04-01

    The blood-brain barrier (BBB) is responsible for the control of solutes' concentration in the brain. Tight junctions and multiple ATP-binding cassette (ABC) and SoLute Carrier (SLC) efflux transporters protect brain cells from xenobiotics, therefore reducing brain exposure to intentionally administered drugs. In epilepsy, polymorphisms and overexpression of efflux transporters genes could be associated with pharmacoresistance. The ontogeny of these efflux transporters should also be addressed because their expression during development may be related to different brain exposure to antiepileptic drugs in the immature brain. We detected statistically significant higher expression of Abcb1b and Slc16a1 genes, and lower expression of Abcb1a and Abcg2 genes between the post-natal day 14 (P14) and the adult rat microvessels. P-gP efflux activity was also shown to be lower in P14 rats when compared with the adults. The P-gP proteins coded by rodent genes Abcb1a and Abcb1b are known to have different substrate affinities. The role of the Abcg2 gene is less clear in pharmacoresistance in epilepsy, nonetheless the coded protein Bcrp is frequently associated with drug resistance. Finally, we observed a higher expression of the Mct1 transporter gene in the P14 rat brain microvessels. Accordingly to our results, we suppose that age may be another factor influencing brain exposure to antiepileptics as a consequence of different expression patterns of efflux transporters between the adult and immature BBB.

  16. Investigation of the quaternary structure of an ABC transporter in living cells using spectrally resolved resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Singh, Deo Raj

    Forster resonance energy transfer (FRET) has become an important tool to study proteins inside living cells. It has been used to explore membrane protein folding and dynamics, determine stoichiometry and geometry of protein complexes, and measure the distance between two molecules. In this dissertation, we use a method based on FRET and optical micro-spectroscopy (OptiMiS) technology, developed in our lab, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of an ABC transporter in living cells. Specifically, the transporter we investigate originates from the pathogen Pseudomonas aeruginosa, which is a Gram-negative bacterium with several virulence factors, lipopolysaccharides being one of them. This pathogen coexpresses two unique forms of lipopolysaccharides on its surface, the A- and B-bands. The A-band polysaccharides, synthesized in the cytoplasm, are translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. In P. aeruginosa, all of the biochemical studies of A-band LPS are concentrated on the stages of the synthesis and ligation of polysaccharides (PSs), leaving the export stage involving ABC transporter unexplored. The mode of PS export through ABC transporters is still unknown. This difficulty is due to the lack of information about sub-unit composition and structure of this bi-component ABC transporter. Using the FRET-OptiMiS combination method developed by our lab, we found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the

  17. Role of the ABC transporter Mdr49 in Hedgehog signaling and germ cell migration.

    PubMed

    Deshpande, Girish; Manry, Diane; Jourjine, Nicholas; Mogila, Vladic; Mozes, Henny; Bialistoky, Tzofia; Gerlitz, Offer; Schedl, Paul

    2016-06-15

    Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet. PMID:27122170

  18. Imaging of P-glycoprotein function and expression to elucidate mechanisms of pharmacoresistance in epilepsy

    PubMed Central

    Löscher, Wolfgang; Langer, Oliver

    2013-01-01

    The issue of pharmacoresistance in epilepsy has received considerable attention in recent years, and a number of plausible hypotheses have been proposed. Of these, the so-called transporter hypothesis is the most extensively researched and documented. This hypothesis assumes that refractory epilepsy is associated with a localised over-expression of drug transporter proteins such as P-glycoprotein (Pgp) in the region of the epileptic focus, which actively extrudes antiepileptic drugs (AEDs) from their intended site of action. However, although this hypothesis has biological plausibility, there is no clinical evidence to support the assertion that AEDs are sufficiently strong substrates for transporter-mediated extrusion from the brain. The use of modern brain imaging techniques to determine Pgp function in patients with refractory epilepsy has started only recently, and may ultimately determine whether increased expression and function of Pgp or other efflux transporters are involved in AED resistance. PMID:20645916

  19. Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

    EPA Science Inventory

    Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

  20. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

    PubMed

    Biswas, E E

    2001-07-27

    Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP > UTP. These

  1. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum.

    PubMed

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-06-15

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s(-1)), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s(-1)), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  2. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

    PubMed Central

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki

    2015-01-01

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  3. Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae.

    PubMed

    Figueira-Mansur, J; Ferreira-Pereira, A; Mansur, J F; Franco, T A; Alvarenga, E S L; Sorgine, M H F; Neves, B C; Melo, A C A; Leal, W S; Masuda, H; Moreira, M F

    2013-12-01

    Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux. PMID:23980723

  4. Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae.

    PubMed

    Figueira-Mansur, J; Ferreira-Pereira, A; Mansur, J F; Franco, T A; Alvarenga, E S L; Sorgine, M H F; Neves, B C; Melo, A C A; Leal, W S; Masuda, H; Moreira, M F

    2013-12-01

    Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux.

  5. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine

    PubMed Central

    Sparreboom, Alex; van Asperen, Judith; Mayer, Ulrich; Schinkel, Alfred H.; Smit, Johan W.; Meijer, Dirk K. F.; Borst, Piet; Nooijen, Willem J.; Beijnen, Jos H.; van Tellingen, Olaf

    1997-01-01

    In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(−/−) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(−/−) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(−/−) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(−/−) mice. The cumulative fecal excretion (0–96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(−/−) mice. Biliary excretion was not significantly different in wt and mdr1a(−/−) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(−/−) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen. PMID:9050899

  6. Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5)

    PubMed Central

    Ravna, Aina W; Sylte, Ingebrigt; Sager, Georg

    2007-01-01

    Background Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette) transporters human P-glycoprotein (ABCB1) and the human MRP5 (ABCC5) are involved in multidrug resistance. Results In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS) of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1)), Ile306 (TMH5), Ile340 (TMH6) and Phe343 (TMH6) may form a binding site, and this is in accordance with previous site directed mutagenesis studies. Conclusion The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5. PMID:17803828

  7. Function and expression of ATP-binding cassette transporters in cultured human Y79 retinoblastoma cells.

    PubMed

    Ishikawa, Yuka; Nagai, Junya; Okada, Yumi; Sato, Koya; Yumoto, Ryoko; Takano, Mikihisa

    2010-01-01

    The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet. PMID:20190417

  8. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    PubMed

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-06-24

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  9. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    PubMed

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-01-01

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation. PMID:26114386

  10. The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters.

    PubMed

    Golin, John; Ambudkar, Suresh V

    2015-05-01

    Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker's yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2. PMID:25886173

  11. The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters

    PubMed Central

    Golin, John; Ambudkar, Suresh V.

    2016-01-01

    Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker’s yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2. PMID:25886173

  12. Integration of in silico and in vitro tools for scaffold optimization during drug discovery: predicting P-glycoprotein efflux.

    PubMed

    Desai, Prashant V; Sawada, Geri A; Watson, Ian A; Raub, Thomas J

    2013-04-01

    In silico tools are regularly utilized for designing and prioritizing compounds to address challenges related to drug metabolism and pharmacokinetics (DMPK) during the process of drug discovery. P-Glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) transporters with broad substrate specificity that plays a significant role in absorption and distribution of drugs that are P-gp substrates. As a result, screening for P-gp transport has now become routine in the drug discovery process. Typically, bidirectional permeability assays are employed to assess in vitro P-gp efflux. In this article, we use P-gp as an example to illustrate a well-validated methodology to effectively integrate in silico and in vitro tools to identify and resolve key barriers during the early stages of drug discovery. A detailed account of development and application of in silico tools such as simple guidelines based on physicochemical properties and more complex quantitative structure-activity relationship (QSAR) models is provided. The tools were developed based on structurally diverse data for more than 2000 compounds generated using a robust P-gp substrate assay over the past several years. Analysis of physicochemical properties revealed a significantly lower proportion (<10%) of P-gp substrates among the compounds with topological polar surface area (TPSA) <60 Å(2) and the most basic cpKa <8. In contrast, this proportion of substrates was greater than 75% for compounds with TPSA >60 Å(2) and the most basic cpKa >8. Among the various QSAR models evaluated to predict P-gp efflux, the Bagging model provided optimum prediction performance for prospective validation based on chronological test sets. Four sequential versions of the model were built with increasing numbers of compounds to train the models as new data became available. Except for the first version with the smallest training set, the QSAR models exhibited robust prediction profiles with positive prediction values (PPV

  13. A subset of annular lipids is linked to the flippase activity of an ABC transporter

    NASA Astrophysics Data System (ADS)

    Bechara, Chérine; Nöll, Anne; Morgner, Nina; Degiacomi, Matteo T.; Tampé, Robert; Robinson, Carol V.

    2015-03-01

    Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.

  14. Arbuscular mycorrhiza affects nickel translocation and expression of ABC transporter and metallothionein genes in Festuca arundinacea.

    PubMed

    Shabani, Leila; Sabzalian, Mohammad R; Mostafavi pour, Sodabeh

    2016-01-01

    Mycorrhizal fungi are key microorganisms for enhancing phytoremediation of soils contaminated with heavy metals. In this study, the effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae (=Glomus mosseae) on physiological and molecular mechanisms involved in the nickel (Ni) tolerance of tall fescue (Festuca arundinacea = Schedonorus arundinaceus) were investigated. Nickel addition had a pronounced negative effect on tall fescue growth and photosynthetic pigment contents, as well as on AMF colonization. Phosphorus content increased markedly in mycorrhizal plants (M) compared to non-inoculated (NM) ones. However, no significant difference was observed in root carbohydrate content between AMF-inoculated and non-inoculated plants. For both M and NM plants, Ni concentrations in shoots and roots increased according to the addition of the metal into soil, but inoculation with F. mosseae led to significantly lower Ni translocation from roots to the aboveground parts compared to non-inoculated plants. ABC transporter and metallothionein transcripts accumulated to considerably higher levels in tall fescue plants colonized by F. mosseae than in the corresponding non-mycorrhizal plants. These results highlight the importance of mycorrhizal colonization in alleviating Ni-induced stress by reducing Ni transport from roots to shoots of tall fescue plants.

  15. ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles.

    PubMed

    Strauss, Anja S; Peters, Sven; Boland, Wilhelm; Burse, Antje

    2013-12-03

    Plant-herbivore interactions dominate the planet's terrestrial ecology. When it comes to host-plant specialization, insects are among the most versatile evolutionary innovators, able to disarm multiple chemical plant defenses. Sequestration is a widespread strategy to detoxify noxious metabolites, frequently for the insect's own benefit against predation. In this study, we describe the broad-spectrum ATP-binding cassette transporter CpMRP of the poplar leaf beetle, Chrysomela populi as the first candidate involved in the sequestration of phytochemicals in insects. CpMRP acts in the defensive glands of the larvae as a pacemaker for the irreversible shuttling of pre-selected metabolites from the hemolymph into defensive secretions. Silencing CpMRP in vivo creates a defenseless phenotype, indicating its role in the secretion process is crucial. In the defensive glands of related leaf beetle species, we identified sequences similar to CpMRP and assume therefore that exocrine gland-based defensive strategies, evolved by these insects to repel their enemies, rely on ABC transporters as a key element. DOI: http://dx.doi.org/10.7554/eLife.01096.001.

  16. The role of ABC and SLC transporters in the pharmacokinetics of dietary and herbal phytochemicals and their interactions with xenobiotics.

    PubMed

    Li, Yan; Lu, Jun; Paxton, James W

    2012-06-01

    There is accumulating evidence that many compounds, known as phytochemicals (PCs), which are derived from dietary plants and herbs, may have a role in combating a number of chronic diseases. Despite many in vitro studies elucidating the mechanism(s) of action of various PCs, there are still reservations with regard to their health benefits in vivo, particularly as there is a paucity of research on their oral bioavailability, their pharmacokinetics, and the concentrations achieved at their site(s) of action. Recently various transporters, including the ATP-binding cassette (ABC) and the solute carrier (SLC) transporters, have been cloned and functional analyses have suggested that they play significant roles in the absorption and disposition of most drugs and PCs. While some SLC transporters facilitate absorption of PCs into the systemic circulation, various efflux pumps, including the ABC transporters, actively transport the PC back into the gastro-intestinal (GI) lumen, thus preventing further penetration into the body. Some ABC transporters also act in concert with Phase 1 and 2 metabolizing enzymes as a defensive barrier in the intestines and liver. If the PC overcomes the defence mechanisms of the gut and the liver, it will enter the systemic circulation and be distributed to the other organs of the body and possible site(s) of action. PCs can usually pass with ease through the pores of the capillaries of organs such as the heart and lungs, but with difficulty into pharmacological sanctuaries, such as the brain, testis, or foetus. Such sanctuaries contain a number of efflux transporters in their protective membrane, which restrict the penetration of xenobiotics, including PCs. The ABC and SLC transporters are also abundantly expressed in the liver and kidney and regulate the excretion of many compounds, including PCs and their metabolites. It is also becoming apparent that there is a complex interplay between various PCs and their ability to modulate the

  17. P-glycoprotein Mediated Efflux Modulators of Plant Origin: A Short Review.

    PubMed

    Silva, Nuno; Salgueiro, Lígia; Fortuna, Ana; Cavaleiro, Carlos

    2016-05-01

    Drug efflux transporters such as P-glycoprotein (P-gp) help maintain cellular homeostasis but are also major contributors to the development of multidrug resistance (MDR) phenomena. Since P-gp was associated with MDR, several compounds showing potential to inhibit this transporter have been identified. Particular attention has been given to natural products, namely those of plant origin, looking for highly effective and safe P-gp inhibitors with little to no interaction with other cellular or metabolic processes. Here we abridge several examples of plant compounds from distinct classes, polyketides, lignans, anthraquinones, coumarins, alkaloids, mono- and sesqui-terpenes, steroids and limonoids, which have shown the ability to modulate in vitro or in vivo the P-gp activity.

  18. Stereoselective Modulation of P-Glycoprotein by Chiral Small Molecules.

    PubMed

    Carocci, Alessia; Catalano, Alessia; Turi, Francesco; Lovece, Angelo; Cavalluzzi, Maria M; Bruno, Claudio; Colabufo, Nicola A; Contino, Marialessandra; Perrone, Maria G; Franchini, Carlo; Lentini, Giovanni

    2016-01-01

    Inhibition of drug efflux pumps such as P-glycoprotein (P-gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N-substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of P-gp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P-gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene-1-yl analogue (R)-2-[(2,3-dichlorophenoxy)methyl]-1-(naphthalen-1-ylmethyl)pyrrolidine) [(R)-7 a] emerged foremost for its potency and stereoselectivity toward P-gp, with the S enantiomer being nearly inactive. The modulation of P-gp by (R)-7 a involved consumption of ATP, thus demonstrating that the compound behaves as a P-gp substrate.

  19. The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

    PubMed Central

    Rivers, Damien M. R.

    2015-01-01

    ABSTRACT Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCE ABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters

  20. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter.

    PubMed Central

    Balan, I; Alarco, A M; Raymond, M

    1997-01-01

    We report the cloning and functional analysis of a third member of the CDR gene family in Candida albicans, named CDR3. This gene codes for an ABC (ATP-binding cassette) transporter of 1,501 amino acids highly homologous to Cdr1p and Cdr2p (56 and 55% amino acid sequence identity, respectively), two transporters involved in fluconazole resistance in C. albicans. The predicted structure of Cdr3p is typical of the PDR/CDR family, with two similar halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six predicted transmembrane segments. Northern analysis showed that CDR3 expression is regulated in a cell-type-specific manner, with low levels of CDR3 mRNA in CAI4 yeast and hyphal cells, high levels in WO-1 opaque cells, and undetectable levels in WO-1 white cells. Disruption of both alleles of CDR3 in CAI4 resulted in no obvious changes in cell morphology, growth rate, or susceptibility to fluconazole. Overexpression of Cdr3p in C. albicans did not result in increased cellular resistance to fluconazole, cycloheximide, and 4-nitroquinoline-N-oxide, which are known substrates for different transporters of the PDR/CDR family. These results indicate that despite a high degree of sequence conservation with C. albicans Cdr1p and Cdr2p, Cdr3p does not appear to be involved in drug resistance, at least to the compounds tested which include the clinically relevant antifungal agent fluconazole. Rather, the high level of Cdr3p expression in WO-1 opaque cells suggests an opaque-phase-associated biological function which remains to be identified. PMID:9393682

  1. Impaired mitochondrial energy production and ABC transporter function-A crucial interconnection in dementing proteopathies of the brain.

    PubMed

    Pahnke, Jens; Fröhlich, Christina; Krohn, Markus; Schumacher, Toni; Paarmann, Kristin

    2013-10-01

    Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. β-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimer's disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

  2. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

    PubMed Central

    2008-01-01

    Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally

  3. Pharmacogenetics of human ABC transporter ABCC11: new insights into apocrine gland growth and metabolite secretion

    PubMed Central

    Ishikawa, Toshihisa; Toyoda, Yu; Yoshiura, Koh-ichiro; Niikawa, Norio

    2013-01-01

    Cell secretion is an important physiological process that ensures smooth metabolic activities and tissue repair as well as growth and immunological functions in the body. Apocrine secretion occurs when the secretory process is accomplished with a partial loss of cell cytoplasm. The secretory materials are contained within secretory vesicles and are released during secretion as cytoplasmic fragments into the glandular lumen or interstitial space. The recent finding that the non-synonymous single nucleotide polymorphisms (SNP) 538G > A (rs17822931; Gly180Arg) in the ABCC11 gene determines the type of earwax in humans has shed light on the novel function of this ABC (ATP-binding cassette) transporter in apocrine glands. The wild-type (Gly180) of ABCC11 is associated with wet-type earwax, axillary osmidrosis, and colostrum secretion from the mammary gland as well as the potential risk of mastopathy. Furthermore, the SNP (538G > A) in the ABCC11 gene is suggested to be a clinical biomarker for the prediction of chemotherapeutic efficacy. The aim of this review article is to provide an overview on the discovery and characterization of genetic polymorphisms in the human ABCC11 gene and to explain the impact of ABCC11 538G > A on the apocrine phenotype as well as the anthropological aspect of this SNP in the ABCC11 gene and patients’ response to nucleoside-based chemotherapy. PMID:23316210

  4. A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence.

    PubMed

    Brown, J S; Gilliland, S M; Holden, D W

    2001-05-01

    Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram-negative pathogens, little is known about how Gram-positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron-dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2- strain was moderately and the pit1-/pit2- strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram-negative bacterial pathogenicity islands. This probable pathogenicity island (PPI-1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.

  5. Tissue distribution and phenobarbital induction of target SLC- and ABC- transporters in cattle.

    PubMed

    Zancanella, V; Giantin, M; Lopparelli, R M; Nebbia, C; Dacasto, M

    2013-08-01

    In veterinary pharmaco-toxicological sciences, few data about uptake and efflux drug transporters (DTs) expression and regulation phenomena have been published. In this study, the tissue distribution and transcriptional modulation of solute carrier (SLC) and ATP-binding cassette (ABC) DTs were investigated in cattle orally administered with phenobarbital (PB) by using a quantitative real-time RT-PCR approach. The criterion for target gene selection was the PB-responsiveness in human and rodent model species. All target DTs were expressed in the liver. Only two of the seven PB-responsive target DTs (SLCO1B3 and SLC10A1) were not constitutively expressed in cattle extra-hepatic tissues. The greatest number of DTs (SLCO2B1, ABCB1, ABCC2, ABCG2) were expressed in intestine and testis, followed by, adrenal gland (SLCO2B1, ABCB1, ABCG2), lung (ABCB1, ABCG2), kidney, and skeletal muscle (ABCG2). PB administration never altered DTs mRNA levels, except for an increase in hepatic ABCC2 mRNA and a down-regulation of renal ABCG2. Altogether, these results confirm only to some extent data obtained in humans and laboratory species; clearly, they should be considered a preliminary step for further molecular investigations about species-differences in DT gene expression and regulation as well as in DT expression and function.

  6. Lysophosphatidylinositol: a novel link between ABC transporters and G-protein-coupled receptors.

    PubMed

    Ruban, Emily L; Ferro, Riccardo; Arifin, Syamsul Ahmad; Falasca, Marco

    2014-10-01

    Lysophosphatidylinositol (LPI) is a well-known bioactive lipid that is able to activate signalling cascades relevant to cell proliferation, migration, survival and tumorigenesis. Our previous work suggested that LPI is involved in cancer progression since it can be released in the medium of Ras-transformed fibroblasts and can function as an autocrine modulator of cell growth. Different research groups have established that LPI is the specific and functional ligand for G-protein-coupled receptor 55 (GPR55) and that this GPR55-LPI axis is able to activate signalling cascades that are relevant for different cell functions. Work in our laboratory has recently unravelled an autocrine loop, by which LPI synthesized by cytosolic phospholipase A₂ (cPLA₂) is pumped out of the cell by ATP-binding cassette (ABC) transporter C1 (ABCC1)/multidrug resistance protein 1 (MRP1), initiating a signalling cascade downstream of GPR55. Our current work suggests that blockade of this pathway may represent a novel strategy to inhibit cancer cell proliferation. PMID:25233417

  7. The Staphylococcus aureus ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst

    PubMed Central

    Coady, Alison; Xu, Min; Phung, Qui; Cheung, Tommy K.; Bakalarski, Corey; Alexander, Mary Kate; Lehar, Sophie M.; Kim, Janice; Park, Summer; Tan, Man-Wah; Nishiyama, Mireille

    2015-01-01

    Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host. PMID:26379037

  8. The Staphylococcus aureus ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst.

    PubMed

    Coady, Alison; Xu, Min; Phung, Qui; Cheung, Tommy K; Bakalarski, Corey; Alexander, Mary Kate; Lehar, Sophie M; Kim, Janice; Park, Summer; Tan, Man-Wah; Nishiyama, Mireille

    2015-01-01

    Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.

  9. A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.

    PubMed

    Shulami, Smadar; Zaide, Galia; Zolotnitsky, Gennady; Langut, Yael; Feld, Geoff; Sonenshein, Abraham L; Shoham, Yuval

    2007-02-01

    Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the -53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 microM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (DeltaCp) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system. PMID:17142383

  10. A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides in Geobacillus stearothermophilus▿

    PubMed Central

    Shulami, Smadar; Zaide, Galia; Zolotnitsky, Gennady; Langut, Yael; Feld, Geoff; Sonenshein, Abraham L.; Shoham, Yuval

    2007-01-01

    Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the −53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 μM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (ΔCp) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system. PMID:17142383

  11. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    SciTech Connect

    Zaja, Roko; Caminada, Daniel; Loncar, Jovica; Fent, Karl; Smital, Tvrtko

    2008-03-01

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 {mu}M doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  12. Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma.

    PubMed

    Huang, Chun-Lin; Ho, Kuo-Chieh

    2007-10-01

    A clone containing a 3903 bp EcoRI-restriction fragment was obtained from a lambda(ZAP) genomic library of loofah witches' broom (LfWB) phytoplasma by plaque hybridization using a PCR fragment as a probe. Sequence analysis revealed that this fragment contained three open reading frames (ORFs). The deduced amino acid sequences of ORF 1 and ORF 2 showed a high homology with the ATP-binding proteins of the ABC transporter system genes of prokaryotes and eukaryotes, and encoded proteins with a molecular mass of 36 and 30 kDa, respectively. Based on amino acid sequence similarity, secondary structure, hydrophilicity and a signal peptide sequence at the N-terminus, we predicted that ORF 3 might encode a specific solute-binding prolipoprotein of the ABC transporter system with a molecular mass of 62 kDa. The cleavage site of this prolipoprotein signal peptide was similar to those of gram-positive bacteria. In addition to nutrient uptake, ABC transporter systems of bacteria also play a role in signal transduction, drug-resistance and perhaps virulence. The possible implications of the system to the survival and the pathogenesis of phytoplasma were discussed.

  13. Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography

    PubMed Central

    Slade, Rozanna L.; Pisaneschi, Federica; Nguyen, Quang-De; Smith, Graham; Carroll, Laurence; Beckley, Alice; Kaliszczak, Maciej A.; Aboagye, Eric O.

    2016-01-01

    Background The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active

  14. Pharmacokinetic Interactions of Herbs with Cytochrome P450 and P-Glycoprotein

    PubMed Central

    Cho, Hyun-Jong

    2015-01-01

    The concurrent use of drugs and herbal products is becoming increasingly prevalent over the last decade. Several herbal products have been known to modulate cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) which are recognized as representative drug metabolizing enzymes and drug transporter, respectively. Thus, a summary of knowledge on the modulation of CYP and P-gp by commonly used herbs can provide robust fundamentals for optimizing CYP and/or P-gp substrate drug-based therapy. Herein, we review ten popular medicinal and/or dietary herbs as perpetrators of CYP- and P-gp-mediated pharmacokinetic herb-drug interactions. The main focus is placed on previous works on the ability of herbal extracts and their phytochemicals to modulate the expression and function of CYP and P-gp in several in vitro and in vivo animal and human systems. PMID:25632290

  15. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain.

    PubMed

    Song, Won-Yong; Yamaki, Tomohiro; Yamaji, Naoki; Ko, Donghwi; Jung, Ki-Hong; Fujii-Kashino, Miho; An, Gynheung; Martinoia, Enrico; Lee, Youngsook; Ma, Jian Feng

    2014-11-01

    Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.

  16. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain

    PubMed Central

    Song, Won-Yong; Yamaki, Tomohiro; Yamaji, Naoki; Ko, Donghwi; Jung, Ki-Hong; Fujii-Kashino, Miho; An, Gynheung; Martinoia, Enrico; Lee, Youngsook; Ma, Jian Feng

    2014-01-01

    Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice. PMID:25331872

  17. Alkamides from Echinacea angustifolia Interact with P-glycoprotein of primary brain capillary endothelial cells isolated from porcine brain blood vessels.

    PubMed

    Mahringer, Anne; Ardjomand-Woelkart, Karin; Bauer, Rudolf; Fricker, Gert; Efferth, Thomas

    2013-03-01

    The blood-brain barrier prevents the passage of toxic compounds from blood circulation into brain tissue. Unfortunately, drugs for the treatment of neurodegenerative diseases, brain tumors, and other diseases also do not cross the blood-brain barrier. In the present investigation, we used isolated porcine brain capillary endothelial cells and a flow cytometric calcein-AM assay to analyze inhibition of P-glycoprotein, a major constituent of the blood-brain barrier. We tested 8 alkamides isolated from Echinacea angustifolia and found that four of them inhibited P-glycoprotein-mediated calcein transport in porcine brain capillary endothelial cells.

  18. Asymmetric localizations of the ABC transporter PaPDR1 trace paths of directional strigolactone transport.

    PubMed

    Sasse, Joëlle; Simon, Sibu; Gübeli, Christian; Liu, Guo-Wei; Cheng, Xi; Friml, Jiří; Bouwmeester, Harro; Martinoia, Enrico; Borghi, Lorenzo

    2015-03-01

    Strigolactones, first discovered as germination stimulants for parasitic weeds [1], are carotenoid-derived phytohormones that play major roles in inhibiting lateral bud outgrowth and promoting plant-mycorrhizal symbiosis [2-4]. Furthermore, strigolactones are involved in the regulation of lateral and adventitious root development, root cell division [5, 6], secondary growth [7], and leaf senescence [8]. Recently, we discovered the strigolactone transporter Petunia axillaris PLEIOTROPIC DRUG RESISTANCE 1 (PaPDR1), which is required for efficient mycorrhizal colonization and inhibition of lateral bud outgrowth [9]. However, how strigolactones are transported through the plant remained unknown. Here we show that PaPDR1 exhibits a cell-type-specific asymmetric localization in different root tissues. In root tips, PaPDR1 is co-expressed with the strigolactone biosynthetic gene DAD1 (CCD8), and it is localized at the apical membrane of root hypodermal cells, presumably mediating the shootward transport of strigolactone. Above the root tip, in the hypodermal passage cells that form gates for the entry of mycorrhizal fungi, PaPDR1 is present in the outer-lateral membrane, compatible with its postulated function as strigolactone exporter from root to soil. Transport studies are in line with our localization studies since (1) a papdr1 mutant displays impaired transport of strigolactones out of the root tip to the shoot as well as into the rhizosphere and (2) DAD1 expression and PIN1/PIN2 levels change in plants deregulated for PDR1 expression, suggestive of variations in endogenous strigolactone contents. In conclusion, our results indicate that the polar localizations of PaPDR1 mediate directional shootward strigolactone transport as well as localized exudation into the soil. PMID:25683808

  19. Tunicamycin depresses P-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in L1210 cells.

    PubMed

    Sereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.

  20. Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

    PubMed Central

    Šereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump. PMID:22174631

  1. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    SciTech Connect

    Filipski, Elisabeth; Berland, Elodie; Ozturk, Narin; Guettier, Catherine; Horst, Gijsbertus T.J. van der; Lévi, Francis; and others

    2014-02-01

    The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F{sub 1} mice. A three-fold 24 h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p < 0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50 mg/kg/day i.v. × 4 days) as a single agent or combined with P-gp inhibitor PSC833 (6.25 mg/kg/day i.p. × 4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40 mg/kg/day × 4 days) and +/− PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p < 0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p < 0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ∼ 60%. In tumor-bearing mice, body weight loss was ∼ halved in the mice on irinotecan or irinotecan–PSC833 combination at ZT15 as compared to ZT3 (p < 0.001). PSC833–irinotecan at ZT15 increased tumor inhibition by ∼ 40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan. - Highlights: • Irinotecan chronotolerance and chronoefficacy change as drug was applied with PSC833. • P-glycoprotein is an important player of the toxicity and efficacy of irinotecan. • Timing should be considered if chemotherapy is performed with a MDR1 inhibitor.

  2. Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

    PubMed Central

    Fricker, G.; Drewe, J.; Huwyler, J.; Gutmann, H.; Beglinger, C.

    1996-01-01

    1. The interaction of cyclosporin A (CyA) with p-glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco-2 cells and an intubation study in healthy volunteers. 2. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco-2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 microM. At higher concentrations permeation increased over-proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 microliter min-1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 microM and a Jmax of 6.5 picomol min-1 per filter. 3. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomycin and a non-immunosuppressive CyA-derivative. All compounds inhibit p-glycoprotein-mediated transport processes. Basolateral to apical permeation of CyA showed a dose-dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco-2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4. Western blot analysis of Caco-2 cells with the monoclonal antibody C219 confirmed the presence of p-glycoprotein in the used cell system. 5. When the absorption of CyA in the gastrointestinal (GI)-tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in

  3. Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level

    PubMed Central

    Smit, Johan W; Duin, Erik; Steen, Herman; Oosting, Roelof; Roggeveld, Jan; Meijer, Dirk K F

    1998-01-01

    In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri-n-butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P-gp substrate doxorubicin (Dox).Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver.We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls.MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general.The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs

  4. AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans.

    PubMed

    Handali, Melody; Roychowdhury, Hridindu; Neupane, Durga P; Yukl, Erik T

    2015-12-11

    Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC). Transcription of the aztD gene is significantly up-regulated under conditions of zinc starvation. Recombinantly expressed AztD purifies with slightly substoichiometric zinc from the periplasm of Escherichia coli and is capable of binding up to three zinc ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound zinc nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a zinc chaperone to AztC and likely functions to maintain zinc homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic zinc trafficking and the function of chaperones in this process.

  5. [Classification models of structure - P-glycoprotein activity of drugs].

    PubMed

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A

    2016-01-01

    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier.

  6. [Classification models of structure - P-glycoprotein activity of drugs].

    PubMed

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A

    2016-01-01

    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier. PMID:27143376

  7. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

    PubMed

    Teng, Yu-Ning; Hsieh, Yow-Wen; Hung, Chin-Chuan; Lin, Hui-Yi

    2015-01-28

    Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 μM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of <1 μM and efficiently inhibited 200 μM verapamil-stimulated ATPase activity, indicating a high affinity of demethoxycurcumin for P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

  8. Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-skin Cancer Effects of Ursolic Acid

    PubMed Central

    Junco, Jacob J.; Mancha, Anna; Malik, Gunjan; Wei, Sung-Jen; Kim, Dae Joon; Liang, Huiyun; Slaga, Thomas J.

    2013-01-01

    Ursolic acid (UA), present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to UA treatment. Herein, skin carcinoma cells (Ca3/7) as compared to skin papilloma cells (MT1/2) displayed more resistance to UA-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in pre-clinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate Rhodamine 123 (Rh123) indicating UA is transported by P-gp. To determine if P-gp inhibition can enhance UA-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil (VRP) or cyclosporin A (CsA). Alternatively, cells were pre-treated with the natural compound resveratrol (RES), a known chemotherapy sensitizer. VRP and RES enhanced the effects of UA in both cell lines, while CsA only did so in Ca3/7 cells. Similarly, VRP inhibited Rh123 efflux in both lines, while CsA only inhibited Rh123 efflux in Ca3/7 cells. RES did not inhibit Rh123 efflux in either line, indicating the synergistic effects of RES and UA are not manifest by inhibition of P-gp-mediated efflux of UA. These results indicate that the anti-skin cancer effects of UA are enhanced with P-gp inhibitors. In addition, RES and UA interact synergistically, but not through inhibition of P-gp. Implications Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen. PMID:24072817

  9. Whole-Transcriptome Survey of the Putative ATP-Binding Cassette (ABC) Transporter Family Genes in the Latex-Producing Laticifers of Hevea brasiliensis

    PubMed Central

    Zhiyi, Nie; Guijuan, Kang; Yu, Li; Longjun, Dai; Rizhong, Zeng

    2015-01-01

    The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 ‘full-size’, 21 ‘half-size’ and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis. PMID:25615936

  10. Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro.

    PubMed

    Subr, V; Sivák, L; Koziolová, E; Braunová, A; Pechar, M; Strohalm, J; Kabešová, M; Ríhová, B; Ulbrich, K; Kovář, M

    2014-08-11

    The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

  11. The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp. PMID:25987565

  12. The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2015-07-01

    P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.

  13. Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.

    PubMed

    Carrier, Isabelle; Julien, Michel; Gros, Philippe

    2003-11-11

    In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.

  14. Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis.

    PubMed

    Ghoshal, Angana; Mukhopadhyay, Sumi; Demine, Rodion; Forgber, Michael; Jarmalavicius, Saulius; Saha, Bibhuti; Sundar, Shyam; Walden, Peter; Mandal, Chhabinath; Mandal, Chitra

    2009-08-01

    We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via alpha2-->6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83-100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins.

  15. P-glycoprotein expression in normal and reactive bone marrows.

    PubMed Central

    Hegewisch-Becker, S.; Fliegner, M.; Tsuruo, T.; Zander, A.; Zeller, W.; Hossfeld, D. K.

    1993-01-01

    The expression of mdr1 gene product P-glycoprotein (P-gp) was investigated in 53 normal and reactive bone marrows by means of immunocytochemistry, using the monoclonal antibody (mAb) C219 and the alkaline phosphatase anti-alkaline phosphatase method. In a limited number of patients, data were confirmed by using the mAb MRK16 or a polymerase chain reaction assay for mdr1 gene expression. There was no history of prior chemotherapy or any malignancy in this group. Bone marrow aspirates were obtained as part of a routine diagnostic programme in bone marrow donors or in patients presenting with a variety of diagnoses such as unexplained gammopathy, fever, anaemia, other changes in peripheral blood smear, rheumatoid arthritis, vasculitis, or urticaria pigmentosa. Morphologically the bone marrow was normal in 23 patients, a megaloblastic erythropoiesis was seen in two patients and unspecific changes were seen in 28 patients. Twenty-seven of 53 samples were found to be positive for P-gp expression with the percentage of positive cells ranging from 2%-80% (mean = 24%). With a cutoff point of 10%, five of 23 normal (22%) and 13 of 28 reactive bone marrows (46%) were considered positive for P-gp expression. There was no obvious correlation between diagnosis or age and P-gp expression. Additional staining for the early surface marker CD-34 was performed in 12 samples, with none of them revealing more than 1% positivity. Since P-gp expression has so far been described only in CD-34 positive bone marrow cells, data suggest that P-gp expression may be reinduced in CD-34 negative cells under conditions which remain to be determined. Images Figure 1 Figure 2 PMID:8094974

  16. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition.

    PubMed

    Filipski, Elisabeth; Berland, Elodie; Ozturk, Narin; Guettier, Catherine; van der Horst, Gijsbertus T J; Lévi, Francis; Okyar, Alper

    2014-02-01

    The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F1 mice. A three-fold 24h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p<0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50mg/kg/day i.v.×4days) as a single agent or combined with P-gp inhibitor PSC833 (6.25mg/kg/day i.p.×4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40mg/kg/day×4days) and +/-PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p<0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p<0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ~60%. In tumor-bearing mice, body weight loss was ~halved in the mice on irinotecan or irinotecan-PSC833 combination at ZT15 as compared to ZT3 (p<0.001). PSC833-irinotecan at ZT15 increased tumor inhibition by ~40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan.

  17. Thyroid Hormone and P-Glycoprotein in Tumor Cells

    PubMed Central

    Davis, Paul J.; Lin, Hung-Yun; Sudha, Thangirala; Mousa, Shaker A.

    2015-01-01

    P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3) at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein. PMID:25866761

  18. The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

    PubMed

    Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

    2016-06-28

    Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. PMID:27033456

  19. In vitro and in vivo evaluations of the P-glycoprotein-mediated efflux of dibenzoylhydrazines.

    PubMed

    Miyata, Ken-Ichi; Nakagawa, Yoshiaki; Kimura, Yasuhisa; Ueda, Kazumitsu; Akamatsu, Miki

    2016-05-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. It actively transports a wide variety of compounds out of cells to protect humans from xenobiotics. Thus, determining whether chemicals are substrates and/or inhibitors of P-gp is important in risk assessments of pharmacokinetic interactions among chemicals because P-gp-mediated transport processes play a significant role in their absorption and disposition. We previously reported that dibenzoylhydrazines (DBHs) such as tebufenozide and methoxyfenozide (agrochemicals) stimulated P-gp ATPase activity. However, it currently remains unclear whether these derivatives are transport substrates of P-gp and inhibit transport of other chemicals by P-gp. In the present study, in order to evaluate the interactions of DBHs with other chemicals in humans, we determined whether DBHs are P-gp transport substrates using both the in vitro bidirectional transport assay and the in vivo study of rats. In the in vivo study, we investigated the influence of P-gp inhibitors on the brain to plasma ratio of methoxyfenozide in rats. We also examined the inhibitory effects of DBHs on quinidine (a P-gp substrate) transport by P-gp in order to ascertain whether these derivatives are inhibitors of P-gp. Based on the results, DBHs were concluded to be weak P-gp transport substrates and moderate P-gp inhibitors. However, the risk of DBHs caused by interaction with other chemicals including drugs was considered to be low by considering the DBHs' potential as the substrates and inhibitors of P-gp as well as their plasma concentrations as long as DBHs are properly used.

  20. Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Andrzejewska, Małgorzata; Ruciński, Marcin; Zabel, Maciej

    2013-04-01

    Multiple drug resistance of cancer cells is multifactorial. A microarray technique may provide information about new candidate genes playing a role in drug resistance. Drug membrane transporters from ABC and SLC families play a main role in this phenomenon. This study demonstrates alterations in ABC and SLC gene expression levels in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant variant of W1 ovarian cancer cell line. Resistant W1 cell lines were derived by stepwise selection of cells in increasing concentration of drugs. Affymetrix GeneChip(®) Human Genome U219 Array Strip was used for hybridizations. Statistical significance was determined by independent sample t-test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by scater plot. Genes up/downregulated more than threefolds were selected and listed. Among ABC genes, seven were upregulated and three were downregulated. Three genes: ABCB1, ABCB4 and ABCG2 were upregulated very significantly (over tenfold). One ABCA8 was significantly downregulated. Among 38 SLC genes, 18 were upregulated, 16 were downregulated and four were up- or downregulated dependent on the cell line. Expression of 10 SLC genes was changed very significantly (over tenfold). Four genes were significantly increased: SLC6A1, SLC9A2, SLC12A1, SLC16A6 and six genes were significantly decreased: SLC2A14, SLC7A3, SLC7A8, SLC7A11, SLC16A14, SLC38A9. Based on the expression profiles, our results provide a preliminary insight into the relationship between drug resistance and expression of membrane transporters involved in drug resistance. Correlation of specific drug transporter with drug resistance requires further analysis.

  1. A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance

    PubMed Central

    Walter, Stephanie; Kahla, Amal; Arunachalam, Chanemoughasoundharam; Perochon, Alexandre; Khan, Mojibur R.; Scofield, Steven R.; Doohan, Fiona M.

    2015-01-01

    The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation

  2. Inhibitory effects of neochamaejasmin B on P-glycoprotein in MDCK-hMDR1 cells and molecular docking of NCB binding in P-glycoprotein.

    PubMed

    Pan, Lanying; Hu, Haihong; Wang, Xiangjun; Yu, Lushan; Jiang, Huidi; Chen, Jianzhong; Lou, Yan; Zeng, Su

    2015-01-01

    Stellera chamaejasme L. (Thymelaeaceae) is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as "Langdu", which is embodied in the Pharmacopoeia of the P.R. China (2010) as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB) on P-glycoprotein (P-gp, ABCB1, MDR1). Rhodamine-123 (R-123) transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1) mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki' values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki', the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one).

  3. P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation.

    PubMed

    Druley, T E; Stein, W D; Ruth, A; Roninson, I B

    2001-04-10

    The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.

  4. The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

    PubMed

    Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

    2016-04-01

    For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea. PMID:26520102

  5. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter - substrate binding protein complex

    PubMed Central

    Nguyen, Phong T.; Li, Qi Wen; Kadaba, Neena S.; Lai, Jeffrey Y.; Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system. PMID:25803078

  6. Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

    PubMed Central

    Young, Rosanna E.B.; Twelkmeyer, Brigitte; Vitiazeva, Varvara; Power, Peter M.; Schweda, Elke K.H.; Hood, Derek W.

    2013-01-01

    Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host–bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component. PMID:24035104

  7. Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

    PubMed

    Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

    2015-03-30

    The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

  8. Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.

    PubMed

    Ghosh, Pratiti; Moitra, Karobi; Maki, Nazli; Dey, Saibal

    2006-06-01

    The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.

  9. Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure

    PubMed Central

    Nicklisch, Sascha C. T.; Rees, Steven D.; McGrath, Aaron P.; Gökirmak, Tufan; Bonito, Lindsay T.; Vermeer, Lydia M.; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

    2016-01-01

    The world’s oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)–100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

  10. Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure.

    PubMed

    Nicklisch, Sascha C T; Rees, Steven D; McGrath, Aaron P; Gökirmak, Tufan; Bonito, Lindsay T; Vermeer, Lydia M; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

    2016-04-01

    The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

  11. Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

    PubMed

    Pawagi, A B; Wang, J; Silverman, M; Reithmeier, R A; Deber, C M

    1994-01-14

    Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization. PMID:7904655

  12. ABC and SLC transporter expression and proton oligopeptide transporter (POT) mediated permeation across the human blood--brain barrier cell line, hCMEC/D3 [corrected].

    PubMed

    Carl, Stephen M; Lindley, David J; Das, Debanjan; Couraud, Pierre O; Weksler, Babette B; Romero, Ignacio; Mowery, Stephanie A; Knipp, Gregory T

    2010-08-01

    Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential

  13. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.

    PubMed

    Macedo, Auricelio A; Silva, Ana P C; Mol, Juliana P S; Costa, Luciana F; Garcia, Luize N N; Araújo, Marcio S; Martins Filho, Olindo A; Paixão, Tatiane A; Santos, Renato L

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  14. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages

    PubMed Central

    Macedo, Auricelio A.; Silva, Ana P. C.; Mol, Juliana P. S.; Costa, Luciana F.; Garcia, Luize N. N.; Araújo, Marcio S.; Martins Filho, Olindo A.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  15. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  16. Functional characterization of the nucleotide binding domain of the Cryptosporidium parvum CpABC4 transporter: an iron-sulfur cluster transporter homolog.

    PubMed

    Benitez, Alvaro J; Arrowood, Michael J; Mead, Jan R

    2009-06-01

    In a previous study, we showed that the Cryptosporidium parvum ATP half-transporter CpABC4 (cgd1_1350) transcript was up-regulated in response to drug treatment with paromomycin and cyclosporine A in an in vitro infection model. CpABC4 may be directly or indirectly involved in the metabolic interactions between host and parasite in response to drug treatment and/or be involved in the intrinsic resistance to chemotherapy. In order to characterize the catalytic site of this transporter, an extended region of the nucleotide-binding domain of CpABC4 (H6-1350NBD) was expressed and purified as an N-terminal hexahistidine-tagged protein in E. coli. The presence of a single tryptophan residue enabled the intrinsic fluorescence to be monitored in response to binding of different compounds. A dose-dependent quenching of the domain's intrinsic fluorescence was observed with its natural substrate, ATP and the fluorescent analogue TNP-ATP. A similar effect was observed with progesterone as well as the flavonoids quercetin and silibinin, previously shown to inhibit parasite development in a cell-based assay. The purified domain also exhibited ATPase activity in the nanomolar range, which further confirmed correct folding and activity of the recombinant domain. The H6-1350NBD serves as a tool to test and design stereospecific inhibitors of the catalytic site, as well as other compounds that bind elsewhere in the domain that may indirectly interact with the catalytic site of the NBD of the CpABC4 transporter.

  17. Polymorphisms in ABC Transporter Genes and Concentrations of Mercury in Newborns – Evidence from Two Mediterranean Birth Cohorts

    PubMed Central

    Llop, Sabrina; Engström, Karin; Ballester, Ferran; Franforte, Elisa; Alhamdow, Ayman; Pisa, Federica; Tratnik, Janja Snoj; Mazej, Datja; Murcia, Mario; Rebagliato, Marisa; Bustamante, Mariona; Sunyer, Jordi; Sofianou-Katsoulis, Αikaterini; Prasouli, Alexia; Antonopoulou, Eleni; Antoniadou, Ioanna; Nakou, Sheena; Barbone, Fabio; Horvat, Milena; Broberg, Karin

    2014-01-01

    Background The genetic background may influence methylmercury (MeHg) metabolism and neurotoxicity. ATP binding cassette (ABC) transporters actively transport various xenobiotics across biological membranes. Objective To investigate the role of ABC polymorphisms as modifiers of prenatal exposure to MeHg. Methods The study population consisted of participants (n = 1651) in two birth cohorts, one in Italy and Greece (PHIME) and the other in Spain (INMA). Women were recruited during pregnancy in Italy and Spain, and during the perinatal period in Greece. Total mercury concentrations were measured in cord blood samples by atomic absorption spectrometry. Maternal fish intake during pregnancy was determined from questionnaires. Polymorphisms (n = 5) in the ABC genes ABCA1, ABCB1, ABCC1 and ABCC2 were analysed in both cohorts. Results ABCB1 rs2032582, ABCC1 rs11075290, and ABCC2 rs2273697 modified the associations between maternal fish intake and cord blood mercury concentrations. The overall interaction coefficient between rs2032582 and log2-transformed fish intake was negative for carriers of GT (β = −0.29, 95%CI −0.47, −0.12) and TT (β = −0.49, 95%CI −0.71, −0.26) versus GG, meaning that for a doubling in fish intake of the mothers, children with the rs2032582 GG genotype accumulated 35% more mercury than children with TT. For rs11075290, the interaction coefficient was negative for carriers of TC (β = −0.12, 95%CI −0.33, 0.09), and TT (β = −0.28, 95%CI −0.51, −0.06) versus CC. For rs2273697, the interaction coefficient was positive when combining GA+AA (β = 0.16, 95%CI 0.01, 0.32) versus GG. Conclusion The ABC transporters appear to play a role in accumulation of MeHg during early development. PMID:24831289

  18. P-glycoprotein inhibitory activity of two phenolic compounds, (-)-syringaresinol and tricin from Sasa borealis.

    PubMed

    Jeong, Yeon Hee; Chung, Soo Yeon; Han, Ah-Reum; Sung, Min Kyung; Jang, Dae Sik; Lee, Jun; Kwon, Youngjoo; Lee, Hwa Jeong; Seo, Eun-Kyoung

    2007-01-01

    (-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR. PMID:17256728

  19. Behavioral Effects and Central Nervous System Levels of the Broadly Available κ-Agonist Hallucinogen Salvinorin A Are Affected by P-Glycoprotein Modulation In Vivo

    PubMed Central

    Caspers, Michael; Lovell, Kimberly M.; Kreek, Mary Jeanne; Prisinzano, Thomas E.

    2012-01-01

    Active blood-brain barrier mechanisms, such as the major efflux transporter P-glycoprotein (mdr1), modulate the in vivo/central nervous system (CNS) effects of many pharmacological agents, whether they are used for nonmedical reasons or in pharmacotherapy. The powerful, widely available hallucinogen salvinorin A (from the plant Salvia divinorum) is a high-efficacy, selective κ-opioid agonist and displays fast-onset behavioral effects (e.g., within 1 min of administration) and relatively short duration of action. In vitro studies suggest that salvinorin A may be a P-glycoprotein substrate; thus, the functional status of P-glycoprotein may influence the behavioral effects of salvinorin A or its residence in CNS after parenteral administration. We therefore studied whether a competing P-glycoprotein substrate (the clinically available agent loperamide; 0.032–0.32 mg/kg) or a selective P-glycoprotein blocker, tariquidar (0.32–3.2 mg/kg) could enhance unconditioned behavioral effects (ptosis and facial relaxation, known to be caused by κ-agonists in nonhuman primates) of salvinorin A, as well as its entry and residence in the CNS, as measured by cerebrospinal fluid sampling. Pretreatment with either loperamide or tariquidar dose-dependently enhanced salvinorin A-induced ptosis, but not facial relaxation. In a control study, loperamide and tariquidar were inactive when given as a pretreatment to ((+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69,593), a κ-agonist known to be a very poor P-glycoprotein substrate. Furthermore, pretreatment with tariquidar (3.2 mg/kg) also enhanced peak levels of salvinorin A in cerebrospinal fluid after intravenous administration. These are the first studies in vivo showing the sensitivity of salvinorin A effects to modulation by the P-glycoprotein transporter, a major functional component of the blood-brain barrier. PMID:22434677

  20. Function of the Caenorhabditis elegans ABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle

    PubMed Central

    Schroeder, Lena K.; Kremer, Susan; Kramer, Maxwell J.; Currie, Erin; Kwan, Elizabeth; Watts, Jennifer L.; Lawrenson, Andrea L.

    2007-01-01

    Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(−) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase–containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(−) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red–stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red–stained fat in pgp-2(−) animals as well as insight into the specialized function of this lysosome-related organelle. PMID:17202409

  1. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis.

    PubMed

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  2. Alkylrhodamines enhance the toxicity of clotrimazole and benzalkonium chloride by interfering with yeast pleiotropic ABC-transporters.

    PubMed

    Knorre, Dmitry A; Besedina, Elizaveta; Karavaeva, Iuliia E; Smirnova, Ekaterina A; Markova, Olga V; Severin, Fedor F

    2016-06-01

    ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this, we studied the effects of several alkylated rhodamines on the drug resistance of yeast Saccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pdr5p substrate-clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had the strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions. PMID:27044313

  3. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis

    PubMed Central

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  4. TetAB(46), a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity

    PubMed Central

    Warburton, Philip J.; Ciric, Lena; Lerner, Avigdor; Seville, Lorna A.; Roberts, Adam P.; Mullany, Peter; Allan, Elaine

    2013-01-01

    Objectives To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient. Methods Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay. Results The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46). Conclusions This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota. PMID:22941900

  5. Deconvoluting the effects of P-glycoprotein on intestinal CYP3A: a major challenge.

    PubMed

    Knight, Beverly; Troutman, Matthew; Thakker, Dhiren R

    2006-10-01

    Metabolism by cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp)-mediated efflux are two important biochemical barriers to drug absorption from the intestine. CYP3A, the most important family of drug-metabolizing enzymes, shares many substrates with the efflux transporter P-gp. Although the individual impact of these two systems on drug disposition is routinely assessed, the effect of both systems acting together during intestinal absorption is difficult to ascertain. Pharmacokinetic theory predicts that the effect of efflux on overall metabolism depends on substrate concentrations relative to the respective kinetic parameters of these processes (i.e. affinities for transport and metabolism, as well as the capacities of these processes). Researchers have published conflicting findings on how efflux affects metabolism. Furthermore, the in vitro parameters that have been used to explain or predict this interation are more relevant for describing overall changes in extraction efficiency of the system (intestinal epithelium), rather than deconvoluting the effect of P-gp on CYP3A-mediated metabolism. Developing a more refined way to understand this interplay and its potential relevance to drug absorption is an important goal, as a large proportion of marketed drugs and many modern drug discovery candidates are known to be affected by one or both of these proteins.

  6. Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

    PubMed Central

    Mohseni, Mahsa; Samadi, Nasser; Ghanbari, Parisa; Yousefi, Bahman; Tabasinezhad, Maryam; Sharifi, Simin; Nazemiyeh, Hossein

    2016-01-01

    Objective(s): Chemoresistance remains the main causes of treatment failure and mortality in cancer patients. There is an urgent need to investigate novel approaches to improve current therapeutic modalities and increase cancer patients’ survival. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in overcoming P-glycoprotein mediated inhibition of apoptosis and induction of cell proliferation in human non-small cell lung carcinoma cells. Materials and Methods: Cell proliferation and apoptosis were assessed using MTT assay and DAPI staining, respectively. P-glycoprotein expression was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively. Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1) to (15±2.6) nM and for vinblastine from (30±5.9) to (5±5.6) nM (P≤0.05). P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001). Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05). Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents. Conclusion: Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression. PMID:27114800

  7. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

    PubMed

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  8. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

    PubMed

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.

  9. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation

    PubMed Central

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10−6 cm/sec, followed by amodiaquine around 20 x 10−6 cm/sec; both mefloquine and artesunate were around 10 x 10−6 cm/sec. Methylene blue was between 2 and 6 x 10−6 cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  10. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

    PubMed Central

    Livnat-Levanon, Nurit; I. Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

  11. Expression of P-glycoprotein in high grade osteosarcomas with special emphasis on chondroblastic subtype.

    PubMed

    Radig, K; Häckel, C; Herting, J; Oda, Y; Mittler, U; Neumann, W; Roessner, A

    1997-02-01

    The development of chemoresistance is one of the major clinical problems in the therapy of malignant bone tumors in childhood. The expression of membrane-bound P-glycoprotein turned out to be an essential factor in the evidence of resistant tumor cells. To investigate the significance of multidrug resistance in the prognosis of highly malignant osteosarcomas, the immunohistologic expression of P-glycoprotein was investigated in the tumor tissue of 52 patients under special consideration of the histologic subtype. The data were compared with the histologic regression grade in the resection specimen and correlated with clinical data. Formalin-fixed, paraffin-embedded tissue and, additionally, fresh frozen material taken from the primary biopsy were stained using monoclonal antibody JSB1. 29 (55%) of the tumors investigated were P-glycoprotein positive. Considering the response to chemotherapy, no conclusion could be drawn regarding P-glycoprotein expression, regression grade in the resection specimens, and the clinical follow-up. P-glycoprotein was detected in only 52% of the non-responders. A positive reaction was also evidenced in 59% of the patients with high chemosensitivity. A comparison of the histologic subtypes yielded a significant result in the chondroblastic osteosarcomas. 11 of 12 cases showed a strong expression of P-glycoprotein. Most of the cases were non-responders, and using Kaplan-Meier live tables, an unfavorable clinical outcome could be demonstrated. Possibly, chondroblastic tumors have a special position among osteosarcomas because of their differentiation. PMID:9065577

  12. Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

    PubMed

    Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. PMID:27602881

  13. Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.

    PubMed

    Parveen, Zahida; Stockner, Thomas; Bentele, Caterina; Pferschy, Sandra; Kraupp, Martin; Freissmuth, Michael; Ecker, Gerhard F; Chiba, Peter

    2011-03-01

    The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs. PMID:21177413

  14. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

    PubMed Central

    Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S.; Bilalis, Dimitrios

    2016-01-01

    Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance. PMID:27104532

  15. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

    PubMed

    Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

    2016-04-20

    Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

  16. P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: A review

    PubMed Central

    Abdallah, Hossam M.; Al-Abd, Ahmed M.; El-Dine, Riham Salah; El-Halawany, Ali M.

    2014-01-01

    Resistance of solid tumors to treatment is significantly attributed to pharmacokinetic reasons at both cellular and multi-cellular levels. Anticancer agent must be bio-available at the site of action in a cytotoxic concentration to exert its proposed activity. P-glycoprotein (P-gp) is a member of the ATP-dependent membrane transport proteins; it is known to pump substrates out of cells in ATP-dependent mechanism. The over-expression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the cytotoxicity of a broad spectrum of antitumor drugs. Accordingly, P-gp inhibitors/blockers are potential enhancer for the cellular bioavailability of several clinically important anticancer drugs such as, anthracyclines, taxanes, vinca alkaloids, and podophyllotoxins. Besides several chemically synthesized P-gp inhibitors/blockers, some naturally occurring compounds and plant extracts were reported for their modulation of multidrug resistance; however, this review will focus only on major classes of naturally occurring inhibitors viz., flavonoids, coumarins, terpenoids, alkaloids and saponins. PMID:25685543

  17. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay

    PubMed Central

    Jouan, Elodie; Le Vée, Marc; Mayati, Abdullah; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug–drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs. PMID:27077878

  18. P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis

    PubMed Central

    Guner, Yigit S.; Franklin, Ashanti L.; Chokshi, Nikunj K.; Castle, Shannon L.; Pontarelli, Elizabeth; Wang, Jin; Wang, Larry; Prasadarao, Nemani V.; Upperman, Jeffrey S.; Grishin, Anatoly V.; Ford, Henri R.

    2014-01-01

    P-glycoprotein (Pgp), a product of the multi-drug resistance gene MDR1a, is a broad specificity efflux ATP cassette transmembrane transporter that is predominantly expressed in epithelial tissues. Because mdr1a−/− mice tend to develop spontaneous colitis in bacteria-dependent manner, Pgp is believed to have a role in protection of the intestinal epithelium from luminal bacteria. Here we demonstrate that levels of Pgp in the small intestine of newborn rodents dramatically increase during breastfeeding, but not during formula feeding (FF). In rats and mice, levels of intestinal Pgp peak on days 3–7 and 1–5 of breastfeeding, respectively. The mdr1a−/− neonatal mice subjected to FF, hypoxia, and hypothermia have significantly higher incidence and pathology, as well as significantly earlier onset of necrotizing enterocolitis (NEC) than congenic wild type mice. Breast-fed mdr1a−/− neonatal mice are also more susceptible to intestinal damage caused by the opportunistic pathogen Cronobacter sakazakii that has been associated with hospital outbreaks of NEC. Breast milk, but not formula, induces Pgp expression in enterocyte cell lines in a dose- and time-dependent manner. High levels of ectopically expressed Pgp protect epithelial cells in vitro from apoptosis induced by C. sakazakii. Taken together, these results show that breast milk-induced expression of Pgp may have a role in the protection of the neonatal intestinal epithelium from injury associated with nascent bacterial colonization. PMID:21788941

  19. Providing a molecular mechanism for P-glycoprotein; why would I bother?

    PubMed

    Callaghan, Richard

    2015-10-01

    It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies.

  20. Comparison of steroid substrates and inhibitors of P-glycoprotein by 3D-QSAR analysis

    NASA Astrophysics Data System (ADS)

    Li, Yan; Wang, Yong-Hua; Yang, Ling; Zhang, Shu-Wei; Liu, Chang-Hou; Yang, Sheng-Li

    2005-01-01

    Steroid derivatives show a complex interaction with P-glycoprotein (Pgp). To determine the essential structural requirements of a series of structurally related and functionally diverse steroids for Pgp-mediated transport or inhibition, a three-dimensional quantitative structure activity relationship study was performed by comparative similarity index analysis modeling. Twelve models have been explored to well correlate the physiochemical features with their biological functions with Pgp on basis of substrate and inhibitor datasets, in which the best predictive model for substrate gave cross-validated q2=0.720, non-cross-validated r2=0.998, standard error of estimate SEE=0.012, F=257.955, and the best predictive model for inhibitor gave q2=0.536, r2=0.950, SEE=1.761 and F=45.800. The predictive ability of all models was validated by a set of compounds that were not included in the training set. The physiochemical similarities and differences of steroids as Pgp substrate and inhibitor, respectively, were analyzed to be helpful in developing new steroid-like compounds.

  1. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay.

    PubMed

    Jouan, Elodie; Le Vée, Marc; Mayati, Abdullah; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug-drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs. PMID:27077878

  2. Pharmacokinetics of single ascending doses of the P-glycoprotein inhibitor tariquidar in healthy subjects

    PubMed Central

    Bauer, M.; Zeitlinger, M.; Todorut, D.; Böhmdorfer, M.; Müller, M.; Langer, O.; Jäger, W.

    2013-01-01

    We assessed the pharmacokinetics (PK), tolerability and safety of tariquidar (TQD), a P-glycoprotein (Pgp) inhibitor, after i.v. administration of single ascending doses. Employed doses were up to 4-fold higher than in previous clinical trials in cancer patients and are capable to inhibit Pgp at the blood-brain barrier (BBB). 15 male healthy volunteers were randomised to receive single i.v. doses of TQD at 4, 6 or 8 mg/kg body weight and underwent blood sampling over 24 h. TQD concentrations were determined in plasma samples with high-performance liquid chromatography-mass spectrometry. No dose limiting toxicities of TQD were observed. Area under the plasma concentration-time curve from start until 24 h after end of infusion (AUC0-n) was positively correlated with administered TQD dose (r=0.8981, p<0.0001). Moreover, we found a positive correlation for volume of distribution at steady state Vss (r=0.7129, p=0.0004) with TQD dose. Dose dependency of Vss points to non-linear PK of TQD, which was in all likelihood caused by transporter saturation at high TQD doses. Acceptable safety and tolerability and dose-linear increases in plasma exposure support future use of TQD at doses up to 8 mg/kg to inhibit Pgp at the human BBB. PMID:23146816

  3. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

    SciTech Connect

    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  4. Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii*

    PubMed Central

    Bottova, Iveta; Hehl, Adrian B.; Štefanić, Saša; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-01-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  5. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Hehl, Adrian B; Stefanić, Sasa; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-06-26

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  6. Interaction of mouse intestinal P-glycoprotein with oral antidiabetic drugs and its inhibitors.

    PubMed

    Kalsi, Harman; Grewal, Ravneet K

    2015-09-01

    Type 2 diabetes (T2DM) is a progressive insulin secretory defect accompanied by resistance to insulin, and thereby making glycemic control a major concern in the treatment of these patients. Oral drug administration, though a popular option for its non-invasiveness, suffer from poor bioavailability. It could be related to the efflux transport of intestinal P-glycoprotein (Pgp). In the present study, we explored the binding interactions of antidiabetic drugs i.e., sulfonylurea drugs (glimepiride, glipizide, glyburide) and rapid acting insulin secretagogues viz., nateglinide, repaglinide and rosiglitazone; and Pgp inhibitors i.e., Generation I (verapamil and tamoxifen), III (tetradrine and tariquidar), and natural inhibitors (fumagillin and piperine) in mouse Pgp model. Our results revealed that fumagillin piperine and verapamil possess maximum interaction energies with Pgp compared to antidiabetic drugs. These observations elucidate the role of fumagillin and piperine as potential natural compounds which could intervene in the efflux action of Pgp in extruding the antidiabetic drugs and may have implications for increasing efficacy of oral antidiabetic therapy. PMID:26548081

  7. Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

    PubMed Central

    Versantvoort, C. H.; Schuurhuis, G. J.; Pinedo, H. M.; Eekman, C. A.; Kuiper, C. M.; Lankelma, J.; Broxterman, H. J.

    1993-01-01

    In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects. Images Figure 1 Figure 6 PMID:8105867

  8. Beneficial effect of tetrandrine on refractory epilepsy via suppressing P-glycoprotein.

    PubMed

    Chen, Yinghui; Xiao, Xia; Wang, Cuicui; Jiang, Huiyuan; Hong, Zhen; Xu, Guoxiong

    2015-01-01

    Patients with refractory epilepsy are resistance to antiepileptic drugs (AEDs). The mechanisms of drug resistance are varied, but one of them is the overexpression of multidrug transporters, such as P-glycoprotein (P-gp), in the brain. Tetrandrine (TTD) is a bis-benzylisoquinoline alkaloid isolated from the root of Stephania tetrandra (S, Moore) and is found to have a favorable effect against multidrug resistance (MDR) in chemotherapy. However, whether TTD affects AEDs in refractory epilepsy is unknown. In this study, we investigated the change in AED treatment efficacy in doxorubicin-induced drug resistant cells after TTD administration. We also examined the effect of TTD on seizure behaviors in the refractory epileptic rats, specifically the expression of MDR1 mRNA and P-gp protein in the cortex and hippocampus of the refractory epileptic rats. Our results demonstrated that TTD decreased cell resistance to phenytoin and valproate. TTD decreased seizure rate and increased the treatment efficacy of AEDs by reducing the expression of P-gp at mRNA and protein levels in vivo. These data support the use of TTD as an adjuvant drug for treating refractory epilepsy. PMID:25233150

  9. Providing a molecular mechanism for P-glycoprotein; why would I bother?

    PubMed Central

    Callaghan, Richard

    2015-01-01

    It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies. PMID:26517914

  10. Providing a molecular mechanism for P-glycoprotein; why would I bother?

    PubMed

    Callaghan, Richard

    2015-10-01

    It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies. PMID:26517914

  11. The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens

    PubMed Central

    Wang, Lulu; Jiang, Rui; Jin, Xiaoling; Liu, Jing; Fan, Shengdi; Quan, Chun-Shan; Ha, Nam-Chul

    2016-01-01

    Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. PMID:27243566

  12. Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

    PubMed Central

    Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

    2012-01-01

    Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

  13. The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens.

    PubMed

    Xu, Yongbin; Guo, Jianyun; Wang, Lulu; Jiang, Rui; Jin, Xiaoling; Liu, Jing; Fan, Shengdi; Quan, Chun-Shan; Ha, Nam-Chul

    2016-01-01

    Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. PMID:27243566

  14. Inhibition mechanism of P-glycoprotein mediated efflux by mPEG-PLA and influence of PLA chain length on P-glycoprotein inhibition activity.

    PubMed

    Li, Wenjing; Li, Xinru; Gao, Yajie; Zhou, Yanxia; Ma, Shujin; Zhao, Yong; Li, Jinwen; Liu, Yan; Wang, Xinglin; Yin, Dongdong

    2014-01-01

    The present study aimed to investigate the effect of monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-PLA) on the activity of P-glycoprotein (P-gp) in Caco-2 cells and further unravel the relationship between PLA chain length in mPEG-PLA and influence on P-gp efflux and the action mechanism. The transport results of rhodamine 123 (R123) across Caco-2 cell monolayers suggested that mPEG-PLA unimers were responsible for its P-gp inhibitory effect. Furthermore, transport studies of R123 revealed that the inhibitory potential of P-gp efflux by mPEG-PLA analogues was strongly correlated with their structural features and showed that the hydrophilic mPEG-PLA copolymers with an intermediate PLA chain length and 10.20 of hydrophilic-lipophilic balance were more effective at inhibiting P-gp efflux in Caco-2 cells. The fluorescence polarization measurement results ruled out the plasma membrane fluidization as a contributor for inhibition of P-gp by mPEG-PLA. Concurrently, mPEG-PLA inhibited neither basal P-gp ATPase (ATP is adenosine triphosphate) activity nor substrate stimulated P-gp ATPase activity, suggesting that mPEG-PLA seemed not to be a substrate of P-gp and a competitive inhibitor. No evident alteration in P-gp surface level was detected by flow cytometry upon exposure of the cells to mPEG-PLA. The depletion of intracellular ATP, which was likely to be a result of partial inhibition of cellular metabolism, was directly correlated with inhibitory potential for P-gp mediated efflux by mPEG-PLA analogues. Hence, intracellular ATP-depletion appeared to be possible explanation to the inhibition mechanism of P-gp by mPEG-PLA. Taken together, the establishment of a relationship between PLA chain length and impact on P-gp efflux activity and interpretation of action mechanism of mPEG-PLA on P-gp are of fundamental importance and will facilitate future development of mPEG-PLA in the drug delivery area.

  15. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

    PubMed Central

    Thong, Shuhua; Ercan, Bilge; Torta, Federico; Fong, Zhen Yang; Wong, Hui Yi Alvina; Wenk, Markus R; Chng, Shu-Sin

    2016-01-01

    In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry. DOI: http://dx.doi.org/10.7554/eLife.19042.001 PMID:27529189

  16. In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

    PubMed Central

    Brewer, Frances K.; Follit, Courtney A.; Vogel, Pia D.

    2014-01-01

    Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. PMID:25270578

  17. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    PubMed

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  18. Understanding polyspecificity within the substrate-binding cavity of the human multidrug resistance P-glycoprotein.

    PubMed

    Martinez, Lorena; Arnaud, Ophélie; Henin, Emilie; Tao, Houchao; Chaptal, Vincent; Doshi, Rupak; Andrieu, Thibault; Dussurgey, Sébastien; Tod, Michel; Di Pietro, Attilio; Zhang, Qinghai; Chang, Geoffrey; Falson, Pierre

    2014-02-01

    Human P-glycoprotein (P-gp) controls drugs bioavailability by pumping structurally unrelated drugs out of cells. The X-ray structure of the mouse P-gp ortholog has been solved, with two SSS enantiomers or one RRR enantiomer of the selenohexapeptide inhibitor QZ59, found within the putative drug-binding pocket (Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL et al. (2009). Science 323, 1718-1722). This offered the first opportunity to localize the well-known H and R drug-binding sites with respect to the QZ59 inhibition mechanisms of Hoechst 33342 and daunorubicin transports, characterized here in cellulo. We found that QZ59-SSS competes efficiently with both substrates, with K(I,app) values of 0.15 and 0.3 μM, which are 13 and 2 times lower, respectively, than the corresponding K(m,app) values. In contrast, QZ59-RRR non-competitively inhibited daunorubicin transport with moderate efficacy (K(I,app) = 1.9 μM); it also displayed a mixed-type inhibition of the Hoechst 33342 transport, resulting from a main non-competitive tendency (K(i2,app) = 1.6 μM) and a limited competitive tendency (K(i1,app) = 5 μM). These results suggest a positional overlap of QZ59 and drugs binding sites: full for the SSS enantiomer and partial for the RRR enantiomer. Crystal structure analysis suggests that the H site overlaps both QZ59-SSS locations while the R site overlaps the most embedded location.

  19. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway.

    PubMed

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M; Nagler, Arnon

    2013-09-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  20. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

    PubMed

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  1. Construction of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette (ABC) transporters and analysis of their growth under stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate since it can survive under multiple stress conditions such as low pH and low temperature. Understanding its survival under stress conditions is important to control this pathogen in food. ABC transporters have been shown...

  2. Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis.

    PubMed Central

    Choudhuri, Baisakhee Saha; Bhakta, Sanjib; Barik, Rajib; Basu, Joyoti; Kundu, Manikuntala; Chakrabarti, Parul

    2002-01-01

    The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters. PMID:12057006

  3. Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

    PubMed Central

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Slotboom, Dirk-Jan

    2015-01-01

    ABSTRACT The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate. IMPORTANCE GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine. PMID:26553850

  4. The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism

    PubMed Central

    van Veen, Hendrik W.; Margolles, Abelardo; Müller, Michael; Higgins, Christopher F.; Konings, Wil N.

    2000-01-01

    The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters. PMID:10835349

  5. Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity.

    PubMed

    Perlin, Michael H; Andrews, Jared; Toh, Su San

    2014-01-01

    Fungi depend heavily on their ability to exploit resources that may become available to them in their myriad of possible lifestyles. Whether this requires simple uptake of sugars as saprobes or competition for host-derived carbohydrates or peptides, fungi must rely on transporters that effectively allow the fungus to accumulate such nutrients from their environments. In other cases, fungi secrete compounds that facilitate their interactions with potential hosts and/or neutralize their competition. Finally, fungi that find themselves on the receiving end of insults, from hosts, competitors, or the overall environment are better served if they can get rid of such toxins or xenobiotics. In this chapter, we update studies on the most ubiquitous transporters, the ABC and MFS superfamilies. In addition, we discuss the importance of subsets of these proteins with particular relevance to plant pathogenic fungi. The availability of ever-increasing numbers of sequenced fungal genomes, combined with high-throughput methods for transcriptome analysis, provides insights previously inaccessible prior to the -omics era. As examples of such broader perspectives, we point to revelations about exploitive use of sugar transporters by plant pathogens, expansion of trichothecene efflux pumps in fungi that do not produce these mycotoxins, and the discovery of a fungal-specific oligopeptide transporter class that, so far, is overrepresented in the plant pathogenic fungi.

  6. The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

    PubMed

    Lee, Miyoung; Choi, Yongwook; Burla, Bo; Kim, Yu-Young; Jeon, Byeongwook; Maeshima, Masayoshi; Yoo, Joo-Yeon; Martinoia, Enrico; Lee, Youngsook

    2008-10-01

    Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

  7. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes.

    PubMed

    Lewis, Daniel R; Miller, Nathan D; Splitt, Bessie L; Wu, Guosheng; Spalding, Edgar P

    2007-06-01

    Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90 degrees reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.

  8. Separating the Roles of Acropetal and Basipetal Auxin Transport on Gravitropism with Mutations in Two Arabidopsis Multidrug Resistance-Like ABC Transporter Genes[W][OA

    PubMed Central

    Lewis, Daniel R.; Miller, Nathan D.; Splitt, Bessie L.; Wu, Guosheng; Spalding, Edgar P.

    2007-01-01

    Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90° reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation. PMID:17557805

  9. Understanding the accumulation of P-glycoprotein substrates within cells: The effect of cholesterol on membrane partitioning.

    PubMed

    Subramanian, Nandhitha; Schumann-Gillett, Alexandra; Mark, Alan E; O'Mara, Megan L

    2016-04-01

    The apparent activity of the multidrug transporter P-glycoprotein (P-gp) is enhanced by the presence of cholesterol. Whether this is due to the direct effect of cholesterol on the activity of P-gp, its effect on the local concentration of substrate in the membrane, or its effect on the rate of entry of the drug into the cell, is unknown. In this study, molecular dynamics simulation techniques coupled with potential of mean force calculations have been used to investigate the role of cholesterol in the movement of four P-gp substrates across a POPC bilayer in the presence or absence of 10% cholesterol. The simulations suggest that the presence of cholesterol lowers the free energy associated with entering the middle of the bilayer in a substrate-specific manner. These findings suggest that P-gp substrates may preferentially accumulate in cholesterol-rich regions of the membrane, which may explain its enhanced transport activity.

  10. Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter

    PubMed Central

    Escudero, Leticia; Mariscal, Vicente

    2015-01-01

    ABSTRACT In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. IMPORTANCE Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular

  11. P-glycoprotein expression and pharmacological modulation in larval stages of Echinococcus granulosus.

    PubMed

    Nicolao, María Celeste; Denegri, Guillermo M; Cárcamo, Juan Guillermo; Cumino, Andrea C

    2014-02-01

    P-glycoprotein (Pgp) is an ATP-dependent transporter involved in the efflux of a wide variety of lipophilic substrates, such as toxins and xenobiotics, out of cells. Pgp expression level is associated with the ineffective therapeutic treatment of cancer cells and microbial pathogens which gives it high clinical importance. Research on these transporters in helminths is limited. This work describes for the first time the Echinococcus granulosus Pgp (Eg-Pgp) expression, in a model cestode parasite and an important human pathogen. Based on calcein efflux assays in the presence of common Pgp modulators, we demonstrated the occurrence of active Eg-Pgp in protoscoleces and metacestodes. Eg-Pgp, which showed a molecular mass of ~130 kDa in western blots, is localized in the suckers and the tegument of control protoscoleces as well as in the subtegument or all parenchymatous cells of protoscoleces treated with Pgp-interfering agents. We also identified five genes encoding Pgp which are constitutively expressed in protoscoleces and metacestodes. We showed that the Eg-pgp1 and Eg-pgp2 transcripts were up-regulated in response to in vitro drug treatment with amiodarone and loperamide, in agreement with the increased polypeptide levels. Finally, in vitro treatment of protoscoleces and metacestodes with trifluoperazine and loperamide was lethal to the parasites. This indicates that both drugs as well as cyclosporine A negatively modulate the E. granulosus Pgp efflux activity, favoring the retention of these drugs in the larval tissue. These events could be associated with the reduction in protoscolex and metacestode viability.

  12. Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state.

    PubMed

    Ramachandra, M; Ambudkar, S V; Chen, D; Hrycyna, C A; Dey, S; Gottesman, M M; Pastan, I

    1998-04-01

    Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

  13. Effects of Curcuma spp. on P-glycoprotein function.

    PubMed

    Ampasavate, Chadarat; Sotanaphun, Uthai; Phattanawasin, Panadda; Piyapolrungroj, Nusara

    2010-06-01

    The effects of Curcuma longa (khamin chan) and Curcuma sp. "khamin-oi" (khamin-oi), as well as isolated major curcuminoids on intestinal P-gp functions were evaluated in vitro. The accumulation of R123 in Caco-2 cells was increased and the R123 efflux ratios were significantly decreased by both Curcuma longa and Curcuma sp. "khamin-oi" extracts, indicating their roles on efflux transporters. The a-b transport of daunorubicin was increased by curcumin, demethoxycurcumin and bisdemethoxycurcumin while the b-a transport was significantly decreased by curcumin and demethoxycurcumin. However, calcein-AM uptake into the human P-gp overexpression cell line, LLC-GA5-COL300, was increased by curcumin and demethoxycurcumin in a concentration-dependent manner but not affected by bisdemethoxycurcumin. These results show that curcumin and demethoxycurcumin could inhibit P-gp but bisdemethoxycurcumin may modulate the function of other efflux transporters such as MRP. Taken together, the information may indicate the impact of Curcuma longa and Curcuma sp. "khamin-oi" on pharmacokinetics of orally administered drugs that are P-gp substrates.

  14. P-glycoprotein expression in canine mammary gland tumours related with myoepithelial cells.

    PubMed

    Kim, N-H; Hwang, Y-H; Im, K-S; Kim, J-H; Chon, S-K; Kim, H-Y; Sur, J-H

    2012-12-01

    P-glycoprotein is influential in chemotherapy-resistance in numerous cancers and has been widely studied in human breast cancer research, but is less studied in canine mammary gland tumour (MGT). The study was to evaluate P-glycoprotein expression and its localisations related with prognostic factors with monoclonal antibody C219, by immunohistochemistry (IHC) of 68 cases of canine malignant (n=54) and benign (n=14) MGT. Additional immunofluorescence (IF) and reverse transcriptase-polymerase chain reaction (RT-PCR) were also performed. There was a novel finding that P-glycoprotein expression with C219 localised at two different cell types: epithelial and myoepithelial cells. Myoepithelial localised tumours were 5 benign (35.5%) and 21 malignant (63.6%), while epithelial localised tumours were 12 cases, all malignant (36.5%). Unlike conventional belief, semi-quantitative evaluation of IHC intensity scores of C219 expression in malignant MGT was related with favourable histopathological parameters. PMID:22554937

  15. P-glycoprotein expression in primary breast cancer detected by immunocytochemistry with two monoclonal antibodies.

    PubMed Central

    Wishart, G. C.; Plumb, J. A.; Going, J. J.; McNicol, A. M.; McArdle, C. S.; Tsuruo, T.; Kaye, S. B.

    1990-01-01

    We have investigated P-glycoprotein (P-gp) expression in samples of primary breast cancer from 29 patients before therapy. We employed immunohistochemical techniques using two monoclonal antibodies (C219 and MRK16) and an indirect alkaline phosphatase method. Heterogeneous expression in epithelial cells was detected with both C219 (21 of 29) and MRK16 (16 of 29). A surprising finding was P-glycoprotein expression in stromal cells with both C219 (26 of 29) and MRK16 (12 of 29). Our results suggest that significant levels of P-glycoprotein expression may be present in breast cancer before exposure to drugs associated with multidrug resistance. Images Figure 1 PMID:1978783

  16. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  17. Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric.

    PubMed

    Lubelski, Jacek; van Merkerk, Ronald; Konings, Wil N; Driessen, Arnold J M

    2006-01-17

    LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs. PMID:16401093

  18. The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

    PubMed

    Kemner, J M; Liang, X; Nester, E W

    1997-04-01

    The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

  19. Molecular Cloning of a 32-Kilodalton Lipoprotein Component of a Novel Iron-Regulated Staphylococcus epidermidis ABC Transporter

    PubMed Central

    Cockayne, Alan; Hill, Philip J.; Powell, Nick B. L.; Bishop, Keith; Sims, Cate; Williams, Paul

    1998-01-01

    Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC

  20. Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and indazole-4,7-dione derivatives towards the Cryptosporidium parvum CpABC3 transporter.

    PubMed

    Zeinyeh, Waël; Xia, Hexue; Lawton, Philippe; Radix, Sylvie; Marminon, Christelle; Nebois, Pascal; Walchshofer, Nadia

    2010-06-01

    The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8a-8f) and indazole-4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian P-glycoproteins.

  1. Fumonisin toxicity in a transgenic mouse model lacking the mdr1a/1b P-glycoprotein genes.

    PubMed

    Sharma; Bhandari; Tsunoda; Riley; Voss; Meredith

    2000-03-01

    The toxicity of fumonisin B(1) (FB(1)) was investigated in male mdr1a/1b double knockout (MDRK) mice, lacking the drug-transporting P-glycoproteins. These transgenic animals are deficient in their blood:brain barrier and accumulate different drugs in brain and other tissues. The MDRK and their wild-type counterparts, FVB mice, were injected subcutaneously with 2.25 mg/kg per day of FB(1) for 5 days and sampled one day after the last treatment in a protocol that has resulted in marked hepatic and renal damage in other strains. FB(1) caused liver enlargement in both FVB and MDRK. Hematological parameters were not affected in either strain. Plasma levels of alanine aminotransferase and aspartate aminotransferase, measures of liver damage, were increased by FB(1) in both FVB and MDRK mice. Histopathological evaluation of liver corroborated this finding. Kidney lesions were induced by FB(1) in both types of mice. Concentrations of free sphingosine and sphinganine increased in liver and kidney of both strains after the FB(1) treatment, although the increase in liver sphingoid bases was half as much in MDRK as compared to FVB. The levels of sphinganine-containing complex sphingolipids were increased in kidney. The levels of sphingosine-containing complex sphingolipids in kidney were unaffected by FB(1) treatment but were significantly lower in control MDRK than in FVB mice. The levels of neurotransmitters and their metabolites were similarly affected in both strains by FB(1), suggesting no influence of disrupted blood:brain barrier on FB(1)-induced neurotoxicity. In both strains, the liver mRNA for tumor necrosis factor alpha was increased; however, the increase was statistically significant only in FVB. It was apparent that mice deficient in P-glycoprotein do not exhibit greater sensitivity to FB(1), the cellular or brain transport of FB(1) appears to be independent of this multidrug transporting system.

  2. Changes in mRNA expression of ABC and SLC transporters in liver and intestines of the adjuvant-induced arthritis rat.

    PubMed

    Uno, Satoshi; Uraki, Misato; Ito, Ayami; Shinozaki, Yuki; Yamada, Ayano; Kawase, Atsushi; Iwaki, Masahiro

    2009-01-01

    In this study, a real-time reverse transcription-polymerase chain reaction was used to determine the effects of adjuvant-induced arthritis (AA) on the amounts of mRNA of 12 types of rat ATP-binding cassette (ABC) and solute carrier (SLC) transporters in the liver and small intestine, 7 (D7) and 21 days (D21) after the injection of adjuvant. There were no significant differences in mRNA levels of ABC and SLC transporters between the livers of AA and control rats on D7, except in the case of Mdr1a. However, levels of Mdr1a, Mrp2 and Oatp SLC transporters were significantly lower in AA than in the control livers on D21. In contrast, the mRNA levels of several ABC and SLC transporters, especially Mrp2, Bcrp, LAT2 and Oatp1a5, were significantly lower in the small intestines of AA rats compared with the controls on D7, though there were no significant differences by D21. The time-dependent alterations in mRNA levels of the pregnane X receptor, but not the constitutive androstane receptor, in the liver and intestine were similar to the changes in mRNA levels of most transporters examined. The present study showed that AA was associated with reduced mRNA expression of several ABC and SLC transporters in the liver and small intestine, but that the time courses of the effects of AA on mRNA expression differed between the liver and small intestine. These results raise the possibility of a functional change of the transporters of liver and intestine in AA rats.

  3. YehZYXW of Escherichia coli Is a Low-Affinity, Non-Osmoregulatory Betaine-Specific ABC Transporter.

    PubMed

    Lang, Shenhui; Cressatti, Marisa; Mendoza, Kris E; Coumoundouros, Chelsea N; Plater, Samantha M; Culham, Doreen E; Kimber, Matthew S; Wood, Janet M

    2015-09-22

    Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.

  4. Dynamics and structural changes induced by ATP and/or substrate binding in the inward-facing conformation state of P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Watanabe, Yurika; Hsu, Wei-Lin; Chiba, Shuntaro; Hayashi, Tomohiko; Furuta, Tadaomi; Sakurai, Minoru

    2013-02-01

    P-glycoprotein (P-gp) is a multidrug transporter that catalyzes the transport of a substrate. To elucidate the underlying mechanism of this type of substrate transport, we performed molecular dynamics (MD) simulations using the X-ray crystal structure of P-gp, which has an inward-facing conformation. Our simulations indicated that the dimerization of the nucleotide binding domains (NBDs) is driven by the binding of ATP to the NBDs and/or the binding of the substrate to a cavity in the transmembrane domains (TMDs). Based on these results, we discuss a role of ATP in the allosteric communication that occurs between the NBDs and the TMDs.

  5. The Effects of Cetirizine on P-glycoprotein Expression and Function In vitro and In situ

    PubMed Central

    Mesgari Abbasi, Mehran; Valizadeh, Hadi; Hamishekar, Hamed; Mohammadnejad, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: P-glycoprotein (P-gp) plays a major role in oral absorption of drugs. Induction or inhibition of P-gp by drugs contributes to variability of its transport activity and often results in clinically relevant drug-drug interactions. The purpose of this study was to investigate the effect of cetirizine, a second generation H1 antihistamine, on P-gp function and expression in vitro and in situ. Methods: The in-vitro rhodamin-123 (Rho123) efflux assay in Caco-2 cells was used to study the effect of cetirizine on P-gp function. Western blot analysis was used for surveying the effect of cetirizine on expression of P-gp in Caco-2 cells. Rat in situ single-pass intestinal permeability technique was used to calculate the intestinal permeability of a known P-gp substrate (digoxin) in the presence of cetirizine. The amounts of digoxin and cetirizine in intestinal perfusion samples were analyzed using a HPLC method. Results: The results showed significant increase in Rho123 uptake (P < 0.05) and also P-gp band intensity decrease in cetirizine-treated cells in vitro. Furthermore the intestinal permeability of digoxin was also increased significantly in the presence of cetirizine (P < 0.01). Conclusion: Therefore it is concluded that cetirizine is a P-gp inhibitor and this should be considered in co administration of cetrizine with other P-gp substrate drugs. Further investigations are required to confirm our results and to determine the mechanism underlying P-gp inhibition by cetirizine. PMID:27123426

  6. P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

    PubMed

    Silva, Renata; Palmeira, Andreia; Carmo, Helena; Barbosa, Daniel José; Gameiro, Mariline; Gomes, Ana; Paiva, Ana Mafalda; Sousa, Emília; Pinto, Madalena; Bastos, Maria de Lourdes; Remião, Fernando

    2015-10-01

    The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

  7. Simotinib as a modulator of P-glycoprotein: substrate, inhibitor, or inducer?

    PubMed

    Huang, Lingling; Shen, Cheng; Chen, Yanfen; Yan, Huiwen; Cheng, Zeneng; Zhu, Qubo

    2016-04-01

    As a new antitumor drug, simotinib hydrochloride is prescribed for prolonged periods, often to patients with comorbidities. Therefore, the risk for developing drug resistance and drug-drug interactions between simotinib and other agents has to be taken into consideration. As P-glycoprotein (P-gp) is an efflux transporter, which plays a significant role in drug resistance and influences the pharmacological properties and toxicities of the drugs it interacts with, the interactions between simotinib and P-gp were investigated. Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular drug concentrations were detected by high-performance liquid chromatography, fluorescence-activated cell sorting and using a fluorescence reader. P-gp ATPase activity was measured using the Pgp-Glo assay, and intracellular pH was assessed using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl. The expression and transcription of P-gp were detected by western blotting and the luciferase assay. Simotinib has no cross-resistance to P-gp substrates, and its efflux rate was independent of either the P-gp expression or the coadministered P-gp substrate. Simotinib reversed chemotherapeutic agent resistance in a short time by increasing the intracellular concentration of the chemotherapeutic agent and blocked rhodamine 123 efflux. Further studies demonstrated that simotinib inhibited P-gp activity by modulating its ATPase activity and the intracellular pH. Although simotinib induced P-gp expression after extended treatment, the induced expression of P-gp had little impact on drug resistance. Simotinib is not a substrate of P-gp. As a modulator, it functions mainly as an inhibitor of P-gp by modulating the intracellular pH and ATPase activity, although it also induces P-gp expression after extended treatment.

  8. P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

    PubMed

    Silva, Renata; Palmeira, Andreia; Carmo, Helena; Barbosa, Daniel José; Gameiro, Mariline; Gomes, Ana; Paiva, Ana Mafalda; Sousa, Emília; Pinto, Madalena; Bastos, Maria de Lourdes; Remião, Fernando

    2015-10-01

    The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation. PMID:25234084

  9. Reversal of P-glycoprotein-mediated multidrug resistance by the novel tetrandrine derivative W6.

    PubMed

    Sun, Hua; Liu, Xiao-Dong; Liu, Qian; Wang, Feng-Peng; Bao, Xiu-Qi; Zhang, Dan

    2015-01-01

    Overexpression of ATP-dependent efflux pump P-glycoprotein (P-gp) is the main cause of multidrug resistance (MDR) and chemotherapy failure in cancer treatment. Inhibition of P-gp-mediated drug efflux is an effective way to overcome cancer drug resistance. The present study investigated the reversal effect of the novel tetrandrine derivative W6 on P-gp-mediated MDR. KBv200, MCF-7/adr and their parental sensitive cell lines KB, MCF-7 were used for reversal study. The intracellular accumulation with P-gp substrates of doxorubicin was determined by flow cytometry. The expression of P-gp and ERK1/2 was investigated by western blot and real-time-PCR (RT-PCR) analysis. ATPase activity of P-gp was performed by P-gp-Glo(TM) assay systems. In comparison with P-gp-negative parental cells, W6 produced a favorable reversal effect in the MDR cells, as determined using the MTT assay. W6 significantly and dose-dependently increased intracellular accumulation of P-gp substrate doxorubicin (DOX) in P-gp overexpressing KBv200 cells, and also inhibited the ATPase activity of P-gp. W6 inhibited P-gp expression in KBv200 cells in a time-dependent manner, but it had no effect on MDR1 expression. In addition, W6 significantly decreased the ERK1/2 activation in KBv200 cells. Our results showed that W6 effectively reversed P-gp-mediated MDR by inhibiting the transport function and expression of P-gp, demonstrating the potential clinical utility of W6. PMID:26235354

  10. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells.

    PubMed

    Lee, Sun-hyo; Kim, Jin-sun; Ravichandran, Kameswaran; Gil, Hyo-Wook; Song, Ho-yeon; Hong, Sae-yong

    2015-01-01

    The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp) induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent manner as measured by DCF-DA. To investigate apoptosis, caspase 3/7 activity was determined. Caspase 3/7 activity was increased dose dependently (25, 50, 100 μg/ml) at 6 h. Autophagosome formation was assessed by measuring LC3- II/LC3-I ratio. The ratio of LC3-II to LC3- I was increased at 2 h (25 μg/ml). Suppression of autophagosome formation increased colistin induced nephrotoxicity. The expression of P-gp and the cell toxicity was determined in colistin with or without dexamethasone (P-gp inducer) and verapamil (selective P-gp inhibitor). Colistin itself suppressed the expression of P-gp. P-gp expression and activity decreased colistin induced nephrotoxicity with dexamethasone treatment. In addition induced P-gp transporter was shown to improve the efflux effect on colistin treated HK2 cell line, which was demonstrated by calcein-AM fluorescence accumulation assay. The increased activity could be blocked by N-acetylcysteine. In conclusion, colistin induces nephrotoxicity by suppressing P-gp. Induction of P-gp could ameliorate colistin induced nephrotoxicity by decreasing apoptosis.

  11. Simotinib as a modulator of P-glycoprotein: substrate, inhibitor, or inducer?

    PubMed

    Huang, Lingling; Shen, Cheng; Chen, Yanfen; Yan, Huiwen; Cheng, Zeneng; Zhu, Qubo

    2016-04-01

    As a new antitumor drug, simotinib hydrochloride is prescribed for prolonged periods, often to patients with comorbidities. Therefore, the risk for developing drug resistance and drug-drug interactions between simotinib and other agents has to be taken into consideration. As P-glycoprotein (P-gp) is an efflux transporter, which plays a significant role in drug resistance and influences the pharmacological properties and toxicities of the drugs it interacts with, the interactions between simotinib and P-gp were investigated. Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular drug concentrations were detected by high-performance liquid chromatography, fluorescence-activated cell sorting and using a fluorescence reader. P-gp ATPase activity was measured using the Pgp-Glo assay, and intracellular pH was assessed using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl. The expression and transcription of P-gp were detected by western blotting and the luciferase assay. Simotinib has no cross-resistance to P-gp substrates, and its efflux rate was independent of either the P-gp expression or the coadministered P-gp substrate. Simotinib reversed chemotherapeutic agent resistance in a short time by increasing the intracellular concentration of the chemotherapeutic agent and blocked rhodamine 123 efflux. Further studies demonstrated that simotinib inhibited P-gp activity by modulating its ATPase activity and the intracellular pH. Although simotinib induced P-gp expression after extended treatment, the induced expression of P-gp had little impact on drug resistance. Simotinib is not a substrate of P-gp. As a modulator, it functions mainly as an inhibitor of P-gp by modulating the intracellular pH and ATPase activity, although it also induces P-gp expression after extended treatment. PMID:26766493

  12. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  13. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    PubMed

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy. PMID:20957096

  14. Bifendate-chalcone hybrids: a new class of potential dual inhibitors of P-glycoprotein and breast cancer resistance protein.

    PubMed

    Gu, Xiaoke; Ren, Zhiguang; Peng, Hui; Peng, Sixun; Zhang, Yihua

    2014-12-12

    We previously described bifendate-chalcone hybrids as potent P-glycoprotein inhibitors. In the present work, we determine whether these compounds could reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance using HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. Results indicated that compounds 8d, 8f, 8g and 8h could significantly enhance mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP drug efflux function. The most active compound 8g exhibited little intrinsic cytotoxicity (IC₅₀>100 μM), and could reverse BCRP-mediated drug resistance independent of decreasing BCRP expression level. Notably, 8g had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. The present findings, together with the previous results, suggest that 8g might be act as dual inhibitors of P-gp and BCRP.

  15. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    PubMed

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

  16. Evaluation of the near infrared compound indocyanine green as a probe substrate of p-glycoprotein.

    PubMed

    Portnoy, Emma; Gurina, Marina; Magdassi, Shlomo; Eyal, Sara

    2012-12-01

    The efflux transporter P-glycoprotein (P-gp) affects the pharmacokinetics of many drugs. Currently used methods for characterization of P-gp's functional activity in vivo involve the use of radiolabeled substrates, are costly, and are technically demanding. Our objective was to evaluate whether the FDA-approved near-infrared compound indocyanine green (ICG) can be used as a probe substrate of P-gp. We also characterized the interaction of ICG with another efflux transporter, the breast cancer resistance protein (BCRP). We evaluated ICG accumulation and transport in MDCK cells overexpressing P-gp or BCRP (MDCK-MDR1 and MDCK-BCRP, respectively) compared to control MDCK cells, in the presence or the absence of transporter inhibitors. In vivo imaging of ICG biodistribution in mice was conducted over 3.5 h using valspodar as the P-gp inhibitor. The EC50 values for ICG accumulation in control MDCK and MDCK-MDR1 cells were 9.0 × 10(-6) ± 5.7 × 10(-7) M and 1.5 × 10(-5) ± 1.1 × 10(-6) M, respectively. The efflux ratio for ICG in MDCK-MDR1 cells was 6.8-fold greater than in control cells. P-gp inhibition attenuated ICG efflux from MDR1-MDCK cells, and their effects in those cells were greater than in control MDCK cells. In contrast, BCRP level of expression or pharmacological inhibition did not significantly affect ICG cellular accumulation. In vivo imaging indicated enhanced cerebral ICG distribution with valspodar (brain - foot area under the concentration-time curves of 3.0 × 10(10), 5.6 × 10(10) and 3.7 × 10(10) h·[p/s/sr]/μW in valspodar-treated mice vs 9.0 × 10(9) and 5.3 × 10(9) h·[p/s/sr]/μW in controls). The findings from this pilot study suggest that near-infrared imaging using ICG as the probe substrate should be further characterized as a methodology for in vivo evaluation of P-gp activity.

  17. Identification of key structural characteristics of Schisandra chinensis lignans involved in P-glycoprotein inhibition.

    PubMed

    Slanina, Jiří; Páchniková, Gabriela; Carnecká, Martina; Porubová Koubíková, Ludmila; Adámková, Lenka; Humpa, Otakar; Smejkal, Karel; Slaninová, Iva

    2014-10-24

    The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure-activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (-)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.

  18. Correction of defective protein kinesis of human P-glycoprotein muta