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Sample records for abc multidrug transporters

  1. [ABC transporter proteins in multidrug resistance of microorganisms].

    PubMed

    Balková, K; Gbelská, Y

    2007-08-01

    The ABC (ATP binding cassette) transporter family includes membrane proteins that can transport a wide variety of substrates across biological membranes. These proteins play an essential role in the protection of cells from toxic compounds/metabolites. Their overexpression which leads to the development of multidrug resistance (MDR) in pathogens and enables cancer cells to survive chemotherapy is of major concern for human health. Mutations in ABC transporters are implicated in a number of Mendelian disorders such as cystic fibrosis, adrenoleukodystrophy and cholesterol and bile transport defects. In microbial cells, several homologues of human ABC transporters were identified. Their further molecular biological study can contribute to better understanding and treatment of MDR or diseases caused by dysfunction of ABC transporter proteins. A review is presented of the state of the art in ABC transporter proteins in both prokaryotic and eucaryotic cells. The role of microbial ABC transporters in the development of drug resistance is analyzed.

  2. A Multidrug ABC Transporter with a Taste for Salt

    PubMed Central

    Gutmann, Daniel A. P.; Venter, Henrietta; Barrera, Nelson P.; Seeger, Markus A.; Woebking, Barbara; Matak-Vinkovic, Dijana; Balakrishnan, Lekshmy; Yao, Yao; U, Edmond C. Y.; Shilling, Richard A.; Robinson, Carol V.; Thorn, Peter; van Veen, Hendrik W.

    2009-01-01

    Background LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H+-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. Methodology/Principal Findings We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H+-Na+-Cl− symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. Conclusions/Significance The observations on H+-Na+-Cl− co-transport substantiate earlier suggestions of H+-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter. PMID:19593434

  3. 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

  4. Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: Evaluation of current strategies

    PubMed Central

    Wu, Chung-Pu; Calcagno, Anna Maria; Ambudkar, Suresh V.

    2008-01-01

    Overexpression of ATP-binding cassette (ABC) drug transporters that actively efflux a variety of amphipathic compounds can cause multidrug resistance (MDR) in cancer cells, which is a major obstacle in the success of cancer chemotherapy. The development of synthetic small molecule compounds or the identification of natural products that block ABC transporter-mediated efflux has been the conventional approach used to combat MDR. The strategy of using chemosensitizers, however, has not been successful in clinical cancer chemotherapy. Therefore, alternative approaches to identify or to synthesize compounds that can induce selective toxicity in cancer cells overexpressing one or more ABC transporters have been undertaken. This review summarizes the recent advances in identifying strategies to restore sensitivity to chemotherapeutics in multidrug resistant cancer cells. PMID:19079736

  5. 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

  6. 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

  7. A silent ABC transporter isolated from Streptomyces rochei F20 induces multidrug resistance.

    PubMed

    Fernández-Moreno, M A; Carbó, L; Cuesta, T; Vallín, C; Malpartida, F

    1998-08-01

    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.

  8. 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

  9. The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB▿ †

    PubMed Central

    Garvey, Mark I.; Piddock, Laura J. V.

    2008-01-01

    One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer. PMID:18362193

  10. 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.

  11. PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones.

    PubMed

    Boncoeur, Emilie; Durmort, Claire; Bernay, Benoît; Ebel, Christine; Di Guilmi, Anne Marie; Croizé, Jacques; Vernet, Thierry; Jault, Jean-Michel

    2012-10-02

    All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.

  12. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

    PubMed

    Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

    2014-10-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

  13. 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

  14. Mitochondrial ABC transporters.

    PubMed

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  15. Plant ABC Transporters

    PubMed Central

    Kang, Joohyun; Park, Jiyoung; Choi, Hyunju; Burla, Bo; Kretzschmar, Tobias; Lee, Youngsook; Martinoia, Enrico

    2011-01-01

    ABC transporters constitute one of the largest protein families found in all living organisms. ABC transporters are driven by ATP hydrolysis and can act as exporters as well as importers. The plant genome encodes for more than 100 ABC transporters, largely exceeding that of other organisms. In Arabidopsis, only 22 out of 130 have been functionally analyzed. They are localized in most membranes of a plant cell such as the plasma membrane, the tonoplast, chloroplasts, mitochondria and peroxisomes and fulfill a multitude of functions. Originally identified as transporters involved in detoxification processes, they have later been shown to be required for organ growth, plant nutrition, plant development, response to abiotic stresses, pathogen resistance and the interaction of the plant with its environment. To fulfill these roles they exhibit different substrate specifies by e.g. depositing surface lipids, accumulating phytate in seeds, and transporting the phytohormones auxin and abscisic acid. The aim of this review is to give an insight into the functions of plant ABC transporters and to show their importance for plant development and survival. PMID:22303277

  16. The ABC-Type Multidrug Resistance Transporter LmrCD Is Responsible for an Extrusion-Based Mechanism of Bile Acid Resistance in Lactococcus lactis▿

    PubMed Central

    Zaidi, Arsalan Haseeb; Bakkes, Patrick J.; Lubelski, Jacek; Agustiandari, Herfita; Kuipers, Oscar P.; Driessen, Arnold J. M.

    2008-01-01

    Upon prolonged exposure to cholate and other toxic compounds, Lactococcus lactis develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in L. lactis and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive L. lactis NZ9000 ΔlmrCD strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of lmrCD upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in L. lactis. PMID:18790870

  17. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

    PubMed Central

    2012-01-01

    Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic

  18. Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

    PubMed

    Panapruksachat, Siribun; Iwatani, Shun; Oura, Takahiro; Vanittanakom, Nongnuch; Chindamporn, Ariya; Niimi, Kyoko; Niimi, Masakazu; Lamping, Erwin; Cannon, Richard D; Kajiwara, Susumu

    2016-07-01

    Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

  19. The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

    PubMed Central

    Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V.

    2008-01-01

    Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone, but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). They inhibited the binding of [125I]-Iodoarylazidoprazosin (IAAP), a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC50 values of 7.3 and 22.6 μM, respectively, but had no effect on the binding of this photoaffinity analog to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of this transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control cells, suggesting that they are substrates of this transporter. Collectively, these data demonstrate for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function. PMID:18065489

  20. MiR-106b~25 cluster regulates multidrug resistance in an ABC transporter-independent manner via downregulation of EP300.

    PubMed

    Hu, Yunhui; Li, Kaiyong; Asaduzzaman, Muhammad; Cuella, Raquel; Shi, Hui; Raguz, Selina; Coombes, Raoul Charles; Zhou, Yuan; Yagüe, Ernesto

    2016-02-01

    MicroRNA (miR)-106b~25 cluster regulates bypass of doxorubicin and γ-radiation induced senescence by downregulation of the E-cadherin transcriptional activator EP300. We asked whether upregulation of miR-106~25 cluster generates cells with a truly multidrug resistant (MDR) phenotype and whether this is due to upregulation of the ATP-binding cassette (ABC) transporter P-glycoprotein. We used minimally transformed mammary epithelial breast cancer cells (MTMECs) in which the miR-106b~25 cluster was experimentally upregulated by lentiviral transfection or in which hairpins targeting either EP300 or E-cadherin mRNAs have been expressed with lentiviruses. We find that overexpression of miR-106b~25 cluster led to the generation of MDR MTMECs (resistant to etoposide, colchicine and paclitaxel). Paclitaxel resistance was also studied after experimental downregulation of EP300 or E-cadherin. However none of these cells overexpressed P-glycoprotein or where able to efflux a fluorescent derivative of paclitaxel, making this phenotype drug-transporter independent. Paclitaxel treatment in MTMECs led to an increase in early apoptotic cells (Annexin V-positive), activation of caspase-9 and increase in the proportion of cells at the G2/M phase of the cell cycle. However, MTMEC overexpressing miR-106b~25 cluster, or with EP300 or E-cadherin downregulated, showed less activation of apoptosis, caspase-9 and caspase-3/-7 activities. Thus, miR-106b~25 cluster controls transporter-independent MDR by apoptosis evasion via downregulation of EP300.

  1. 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

  2. The ABC transporter gene family of Daphnia pulex

    PubMed Central

    Sturm, Armin; Cunningham, Phil; Dean, Michael

    2009-01-01

    Background The large gene superfamily of ABC (ATP-binding cassette) transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex, providing for the first time information on ABC proteins in crustacea, a primarily aquatic arthropod subphylum of high ecological and economical importance. Results We identified 64 ABC proteins in the Daphnia genome, which possesses members of all current ABC subfamilies A to H. To unravel phylogenetic relationships, ABC proteins of Daphnia were compared to those from yeast, worm, fruit fly and human. A high conservation of Daphnia of ABC transporters was observed for proteins involved in fundamental cellular processes, including the mitochondrial half transporters of the ABCB subfamily, which function in iron metabolism and transport of Fe/S protein precursors, and the members of subfamilies ABCD, ABCE and ABCF, which have roles in very long chain fatty acid transport, initiation of gene transcription and protein translation, respectively. A number of Daphnia proteins showed one-to-one orthologous relationships to Drosophila ABC proteins including the sulfonyl urea receptor (SUR), the ecdysone transporter ET23, and the eye pigment precursor transporter scarlet. As the fruit fly, Daphnia lacked homologues to the TAP protein, which plays a role in antigene processing, and the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride channel. Daphnia showed two proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and six proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCC subfamily). However, lineage specific gene duplications in the ABCB and ABCC subfamilies complicated the inference of function. A

  3. Effluxing ABC Transporters in Human Corneal Epithelium

    PubMed Central

    Vellonen, Kati-Sisko; Mannermaa, Eliisa; Turner, Helen; Häkli, Marika; Wolosin, J. Mario; Tervo, Timo; Honkakoski, Paavo; Urtti, Arto

    2010-01-01

    ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1–6 (MRP1–6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. PMID:19623615

  4. ABC transporters and the blood-brain barrier.

    PubMed

    Begley, David J

    2004-01-01

    The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) form a very effective barrier to the free diffusion of many polar solutes into the brain. Many metabolites that are polar have their brain entry facilitated by specific inwardly-directed transport mechanisms. In general the more lipid soluble a molecule or drug is, the more readily it will tend to partition into brain tissue. However, a very significant number of lipid soluble molecules, among them many useful therapeutic drugs have lower brain permeability than would be predicted from a determination of their lipid solubility. These molecules are substrates for the ABC efflux transporters which are present in the BBB and BCSB and the activity of these transporters very efficiently removes the drug from the CNS, thus limiting brain uptake. P-glycoprotein (Pgp) was the first of these ABC transporters to be described, followed by the multidrug resistance-associated proteins (MRP) and more recently breast cancer resistance protein (BCRP). All are expressed in the BBB and BCSFB and combine to reduce the brain penetration of many drugs. This phenomenon of "multidrug resistance" is a major hurdle when it comes to the delivery of therapeutics to the brain, not to mention the problem of cancer chemotherapy in general. Therefore, the development of strategies for bypassing the influence of these ABC transporters and for the design of effective drugs that are not substrates and the development of inhibitors for the ABC transporters becomes a high imperative for the pharmaceutical industry.

  5. ABC transporters in the CNS - an inventory.

    PubMed

    Hartz, A M S; Bauer, B

    2011-04-01

    In the present review we provide a summary of ATP-binding cassette (ABC) transporters in the central nervous system (CNS). Our review is focused on transporters of the ABC A, B, C, D, and G families that have been detected in the cells of the neurovascular unit/blood-brain barrier including brain capillary endothelial cells, pericytes, astrocytes, and neurons, as well as in other brain cells, such as microglia, oligodendrocytes, and choroid plexus epithelial cells. In this review, we provide an overview, organized by ABC family, of transporter expression, localization, and function. We summarize recent findings on ABC transporter regulation in the CNS and address the role of ABC transporters in CNS diseases including brain cancer, seizures/epilepsy, and Alzheimer's disease. Finally, we discuss new therapeutic strategies focused on ABC transporters in CNS disease.

  6. 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.

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

    SciTech Connect

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

    2016-07-27

    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 groupP1), with unit-cell parametersa= 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.

  8. A Drosophila ABC Transporter Regulates Lifespan

    PubMed Central

    Huang, He; Lu-Bo, Ying; Haddad, Gabriel G.

    2014-01-01

    MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling. PMID:25474322

  9. 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

  10. 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

  11. The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2.

    PubMed

    Shukla, Suneet; Robey, Robert W; Bates, Susan E; Ambudkar, Suresh V

    2006-07-25

    The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.

  12. AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli.

    PubMed

    Horiyama, Tsukasa; Nishino, Kunihiko

    2014-01-01

    Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin.

  13. Structural and mechanistic diversity of multidrug transporters.

    PubMed

    Mousa, Jarrod J; Bruner, Steven D

    2016-10-26

    Covering: 2009 to mid 2016Multidrug transporters are common and prevalent in all orders of life, having diverse functions from the removal of toxins, resistance to cytotoxins, and the transport of specific eluents. In addition, multidrug transporters pose a significant threat to modern medicine. Able to transport structurally diverse small molecule drugs, these transporters are implicated in antibiotic resistant strains of bacteria, as well as chemotherapeutic-resistance cancer cells. Although important in such resistance, a relatively small number of multidrug transporters have been structurally characterized, primarily due to the difficulty in purifying and crystallizing active membrane proteins and protein complexes. This review will cover recent structural breakthroughs in the past six years that have led to increased knowledge of the mechanisms of multidrug transporter chemistry, and the role of these transporters in exporting secondary metabolites.

  14. The role of multidrug transporters in drug availability, metabolism and toxicity.

    PubMed

    Bodó, Adrienn; Bakos, Eva; Szeri, Flóra; Váradi, András; Sarkadi, Balázs

    2003-04-11

    Multidrug resistance is frequently observed when treating cancer patients with chemotherapeutic agents. A variety of ATP binding cassette (ABC) transporters, localized in the cell membrane, cause this phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells. However, the major physiological role of the multidrug transporters is the protection of our cells and tissues against xenobiotics, and these transporters play a key role in drug availability, metabolism and toxicity. Three major groups of ABC transporters are involved in multidrug resistance: the classical P-glycoprotein MDR1, the multidrug resistance associated proteins (MRP1, MRP2, and probably MRP3, MRP4 and MRP5), and the ABCG2 protein, an ABC half-transporter. All these proteins were shown to catalyze an ATP-dependent active transport of chemically unrelated compounds. 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. By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved. Oral bioavailability, entering the blood-brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins. Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics. Here we show that by using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents. We focus on the characterisation of the MRP1 and MRP3 proteins, their relevance in chemoresistance of cancer and in

  15. Multidrug transporters as drug targets.

    PubMed

    Liang, X-J; Aszalos, A

    2006-08-01

    Transport molecules can significantly affect the pharmacodynamics and pharmacokinetics of drugs. An important transport molecule, the 170 kDa P-glycoprotein (Pgp), is constitutively expressed at several organ sites in the human body. Pgp is expressed at the blood-brain barrier, in the kidneys, liver, intestines and in certain T cells. Other transporters such as the multidrug resistance protein 1 (MRP1) and MRP2 also contribute to drug distribution in the human body, although to a lesser extent than Pgp. These three transporters, and especially Pgp, are often targets of drugs. Pgp can be an intentional or unintentional target. It is directly targeted when one wants to block its function by a modifier drug so that another drug, also a substrate of Pgp, can penetrate the cell membrane, which would otherwise be impermeable. Unintentional targeting occurs when several drugs are administered to a patient and as a consequence, the physiological function of Pgp is blocked at different organ sites. Like Pgp, MRP1 also has the capacity to mediate transport of many drugs and other compounds. MRP1 has a protective role in preventing accumulation of toxic compounds and drugs in epithelial tissue covering the choroid plexus/cerebrospinal fluid compartment, oral epithelium, sertoli cells, intesticular tubules and urinary collecting duct cells. MRP2 primarily transports weakly basic drugs and bilirubin from the liver to bile. Most compounds that efficiently block Pgp have only low affinity for MRP1 and MRP2. There are only a few effective and specific MRP inhibitors available. Drug targeting of these transporters may play a role in cancer chemotherapy and in the pharmacokinetics of substrate drugs.

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

    PubMed

    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

  17. 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

  18. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

    2013-06-01

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

  19. The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum.

    PubMed

    Weber, Stefan S; Kovalchuk, Andriy; Bovenberg, Roel A L; Driessen, Arnold J M

    2012-11-01

    The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid.

  20. Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters.

    PubMed

    Tannert, Astrid; Pohl, Antje; Pomorski, Thomas; Herrmann, Andreas

    2003-09-01

    Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids. Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP. Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP. A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells. Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells. This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis. The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed.

  1. 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

  2. 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

  3. Genome-wide identification of ATP-binding cassette (ABC) transporters and conservation of their xenobiotic transporter function in the monogonont rotifer (Brachionus koreanus).

    PubMed

    Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Young Hwan; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

    2017-03-01

    The ATP-binding cassette (ABC) transporter family is one of the largest gene family in animals, and members of this family are known to be involved in various biological processes due to their ability to transport a wide range of substrates across membranes using ATP cleavage-derived energy. We identified 61 ABC transporters in the genome of the monogonont rotifer Brachionus koreanus, and classified these into eight distinct subfamilies (A-H) by phylogenetic analysis. ABC transporters in the rotifer B. koreanus are comprised of 11 ABCA genes, 19 ABCB genes, 14 ABCC genes, 3 ABCD genes, 1 ABCE gene, 3 ABCF genes, 8 ABCG genes, and 2 ABCH genes. Extensive gene duplication and loss events in synteny were observed in several subfamilies. In particular, massive gene duplications of P-glycoproteins (P-gps), multidrug resistance proteins (MRPs), and Bk-Abcg-like proteins were observed. The ability of these B. koreanus proteins to function as multixenobiotic resistance (MXR) ABC transporters was validated using specific fluorescence substrates/inhibitors. The ABC transporter superfamily members identified in this study will be useful in future toxicological studies, and will facilitate comparative studies of the evolution of the ABC transporter superfamily in invertebrates.

  4. Genome-wide analysis of the ATP-binding cassette (ABC) transporter gene family in the silkworm, Bombyx mori.

    PubMed

    Xie, Xiaodong; Cheng, Tingcai; Wang, Genhong; Duan, Jun; Niu, Weihuan; Xia, Qingyou

    2012-07-01

    The ATP-binding cassette (ABC) superfamily is a larger protein family with diverse physiological functions in all kingdoms of life. We identified 53 ABC transporters in the silkworm genome, and classified them into eight subfamilies (A-H). Comparative genome analysis revealed that the silkworm has an expanded ABCC subfamily with more members than Drosophila melanogaster, Caenorhabditis elegans, or Homo sapiens. Phylogenetic analysis showed that the ABCE and ABCF genes were highly conserved in the silkworm, indicating possible involvement in fundamental biological processes. Five multidrug resistance-related genes in the ABCB subfamily and two multidrug resistance-associated-related genes in the ABCC subfamily indicated involvement in biochemical defense. Genetic variation analysis revealed four ABC genes that might be evolving under positive selection. Moreover, the silkworm ABCC4 gene might be important for silkworm domestication. Microarray analysis showed that the silkworm ABC genes had distinct expression patterns in different tissues on day 3 of the fifth instar. These results might provide new insights for further functional studies on the ABC genes in the silkworm genome.

  5. 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.

  6. Inhibition of ABC Transporters Abolishes Antimony Resistance in Leishmania Infection▿

    PubMed Central

    Mookerjee Basu, Jayati; Mookerjee, Ananda; Banerjee, Rajdeep; Saha, Manik; Singh, Subhankar; Naskar, Ksudiram; Tripathy, Gayetri; Sinha, Prabhat K.; Pandey, Krishna; Sundar, Shyam; Bimal, Sanjeev; Das, Pradip K.; Choudhuri, Soumitra K.; Roy, Syamal

    2008-01-01

    The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sbr) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sbr L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer. PMID:18056276

  7. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.

    PubMed

    Sarkadi, Balázs; Homolya, László; Szakács, Gergely; Váradi, András

    2006-10-01

    In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

  8. Coupled ATPase-adenylate kinase activity in ABC transporters

    PubMed Central

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  9. Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters.

    PubMed

    Infed, Nacera; Smits, Sander H J; Dittrich, Torsten; Braun, Manfred; Driessen, Arnold J M; Hanekop, Nils; Schmitt, Lutz

    2013-03-01

    The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC(50) values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.

  10. MFS multidrug transporters in pathogenic fungi: do they have real clinical impact?

    PubMed Central

    Costa, Catarina; Dias, Paulo J.; Sá-Correia, Isabel; Teixeira, Miguel C.

    2014-01-01

    Infections caused by opportunistic fungal pathogens have reached concerning numbers due to the increase of the immunocrompromised human population and to the development of antifungal resistance. This resistance is often attributed to the action of multidrug efflux pumps, belonging to the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). Although many studies have focused on the role of ABC multidrug efflux transporters, little is still known on the part played by the Drug:H+ Antiporter (DHA) family of the MFS in this context. This review summarizes current knowledge on the role in antifungal drug resistance, mode of action and phylogenetic relations of DHA transporters, from the model yeast S. cerevisiae to pathogenic yeasts and filamentous fungi. Through the compilation of the predicted DHA transporters in the medically relevant Candida albicans, C. glabrata, C. parapsilosis, C. lusitaniae, C. tropicalis, C. guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus species, the fact that only 5% of the DHA transporters from these organisms have been characterized so far is evidenced. The role of these transporters in antifungal drug resistance and in pathogen-host interaction is described and their clinical relevance discussed. Given the knowledge gathered for these few DHA transporters, the need to carry out a systematic characterization of the DHA multidrug efflux pumps in fungal pathogens, with emphasis on their clinical relevance, is highlighted. PMID:24904431

  11. Differential Contributions of Five ABC Transporters to Mutidrug Resistance, Antioxidion and Virulence of Beauveria bassiana, an Entomopathogenic Fungus

    PubMed Central

    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. PMID:23596534

  12. Regulation of ABC Transporters at the Blood-Brain Barrier

    PubMed Central

    Miller, David S.

    2015-01-01

    ATP Binding Cassette (ABC) transporters at the blood-brain barrier function as ATP-driven xenobiotic efflux pumps and limit delivery of small molecule drugs to the brain. Here I review recent progress in understanding the regulation of the expression and transport activity of these transporters and comment on how this new information might aid in improving drug delivery to the brain. PMID:25670036

  13. Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters

    PubMed Central

    Shimokawa, Yoshihiko; Shibata, Masakazu; Hashizume, Kenta; Hamasako, Yusuke; Ohzone, Yoshihiro; Kashiyama, Eiji; Umehara, Ken

    2016-01-01

    Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro. Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 μmol/liter, respectively; M2, 7.80 and 6.02 μmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters. PMID:27021329

  14. Evaluation of current methods used to analyze the expression profiles of ABC transporters yields an improved drug-discovery database

    PubMed Central

    Orina, Josiah N.; Calcagno, Anna Maria; Wu, Chung-Pu; Varma, Sudhir; Shih, Joanna; Lin, Min; Eichler, Gabriel; Weinstein, John N.; Pommier, Yves; Ambudkar, Suresh V.; Gottesman, Michael M.; Gillet, Jean-Pierre

    2009-01-01

    The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anti-cancer drug and can develop by multiple mechanisms. These mechanisms can act individually or synergistically, leading to multidrug resistance (MDR), in which the cell becomes resistant to a variety of structurally and mechanistically unrelated drugs in addition to the drug initially administered. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been successful. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing highly homologous genes from small amounts of tissue is fundamental to achieving any significant enhancement in our understanding of multidrug resistance mechanisms and could lead to treatments designed to circumvent it. In this study, we use a previously established database that allows the identification of lead compounds in the early stages of drug discovery that are not ABC transporter substrates. We believe this can serve as a model for appraising the accuracy and sensitivity of current methods used to analyze the expression profiles of ABC transporters. We found two platforms to be superior methods for the analysis of expression profiles of highly homologous gene superfamilies. This study also led to an improved database by revealing previously unidentified substrates for ABCB1, ABCC1 and ABCG2, transporters that contribute to multidrug resistance. PMID:19584229

  15. ABC-transporters: implications on drug resistance from microorganisms to human cancers.

    PubMed

    Lage, Hermann

    2003-09-01

    Resistance to chemotherapy is a common clinical problem in patients with infectious diseases as well as in patients with cancer. During treatment of infections or malignant tumors, the drug targets of prokaryotic or eukaryotic microorganisms and neoplastic cells are often found to be refractory to a variety of drugs that have different structures and functions. This phenomenon has been termed multidrug resistance (MDR). The mechanisms leading to MDR are frequently caused by trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC) transporters. There is an urgent need to understand the structure-function relationships of these efflux pumps that underlie their transport mechanism and drug selectivity. This knowledge may allow the rational design of new drugs that can inhibit or circumvent the activity of these MDR transport molecules. Furthermore, the development of such chemosensitizing agents would help us learn more about the physiological functions and substrates of these pump proteins. This review will discuss the current state of knowledge of the functional and structural similarities among ABC-transporters in prokaryotic and eukaryotic cells and their impact on MDR.

  16. 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.

  17. Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance*

    PubMed Central

    Mendoza-Cózatl, David G.; Zhai, Zhiyang; Jobe, Timothy O.; Akmakjian, Garo Z.; Song, Won-Yong; Limbo, Oliver; Russell, Matthew R.; Kozlovskyy, Volodymyr I.; Martinoia, Enrico; Vatamaniuk, Olena K.; Russell, Paul; Schroeder, Julian I.

    2010-01-01

    Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates 35S-PC2 uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms. PMID:20937798

  18. Structure of a Type-1 Secretion System ABC Transporter.

    PubMed

    Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

    2017-03-07

    Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access.

  19. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter.

    PubMed

    Yang, Nuan; Driessen, Arnold J M

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

  20. Exploring conformational equilibria of a heterodimeric ABC transporter

    PubMed Central

    Timachi, M Hadi; Hutter, Cedric AJ; Hohl, Michael; Assafa, Tufa; Böhm, Simon; Mittal, Anshumali; Seeger, Markus A; Bordignon, Enrica

    2017-01-01

    ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis. DOI: http://dx.doi.org/10.7554/eLife.20236.001 PMID:28051765

  1. 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

  2. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors.

    PubMed Central

    Sanglard, D; Ischer, F; Monod, M; Bille, J

    1996-01-01

    Some Candida albicans isolates from AIDS patients with oropharyngeal candidiasis are becoming resistant to the azole antifungal agent fluconazole after prolonged treatment with this compound. Most of the C. albicans isolates resistant to fluconazole fail to accumulate this antifungal agent, and this has been considered a cause of resistance. This phenomenon was shown to be linked to an increase in the amounts of mRNA of a C. albicans ABC (ATP-binding cassette) transporter gene called CDR1 and of a gene conferring benomyl resistance (BENr), the product of which belongs to the class of major facilitator multidrug efflux transporters (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). To analyze the roles of these multidrug transporters in the efflux of azole antifungal agents, we constructed C. albicans mutants with single and double deletion mutations of the corresponding genes. The mutants were tested for their susceptibilities to these antifungal agents. Our results indicated that the delta cdr1 C. albicans mutant was hypersusceptible to the azole derivatives fluconazole, itraconazole, and ketoconazole, thus showing that the ABC transporter Cdr1 can use these compounds as substrates. The delta cdr1 mutant was also hypersusceptible to other antifungal agents (terbinafine and amorolfine) and to different metabolic inhibitors (cycloheximide, brefeldin A, and fluphenazine). The same mutant was slightly more susceptible than the wild type to nocodazole, cerulenin, and crystal violet but not to amphotericin B, nikkomycin Z, flucytosine, or pradimicin. In contrast, the delta ben mutant was rendered more susceptible only to the mutagen 4-nitroquinoline-N-oxide. However, this mutation increased the susceptibilities of the cells to cycloheximide and cerulenin when the mutation was constructed in a delta cdr1 background. The assay used in the present study could be implemented with new antifungal

  3. A primer on the mechanics of P-glycoprotein the multidrug transporter.

    PubMed

    Hennessy, M; Spiers, J P

    2007-01-01

    P-glycoprotein (P-gp) the multidrug transporter is a well-characterised member of the super-family of ATP-binding cassette (ABC) transporters, and mediates the clearance of xenotoxins against steep concentration gradients at the expense of ATP hydrolysis. The primary function of this protein is to prevent the uptake of toxic compounds from the gut into the body, and to protect vital structures such as the brain, cerebrospinal fluid, testis, foetus and bone marrow against toxins. Although P-gp transports a wide range of compounds, which is advantageous, it can also be a disadvantage and may interfere with the delivery of drugs to target tissues resulting in multidrug resistance. In the present review: (i) we consider our current understanding of the structure of P-glycoprotein, (ii) discuss substrate binding and its coupling to ATPase activity, (iii) provide insight into key features which define P-glycoprotein substrates/inhibitors and the ability to predict potential substrates in silico, (iv) provide an overview of existing models of pump function and (v) present emerging concepts into the regulation of P-glycoprotein expression, with particular reference to multidrug resistance.

  4. 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.

  5. Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer.

    PubMed

    Chen, Yi; Bieber, Marcia M; Teng, Nelson N H

    2014-08-01

    A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused by active drug efflux mediated by ATP-binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

  6. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Merino, Gracia; Jonker, Johan W; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2007-02-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

  7. bmr3, a third multidrug transporter gene of Bacillus subtilis.

    PubMed Central

    Ohki, R; Murata, M

    1997-01-01

    A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase. PMID:9023234

  8. The multidrug-resistance transporter ABCB5 is expressed in human placenta.

    PubMed

    Volpicelli, Elgida R; Lezcano, Cecilia; Zhan, Qian; Girouard, Sasha D; Kindelberger, David W; Frank, Markus H; Frank, Natasha Y; Crum, Christopher P; Murphy, George F

    2014-01-01

    ATP-binding cassette (ABC) transporters in placenta protectively transport drugs and xenobiotics. ABCB5 [subfamily B (MDR/TAP)] is a novel ABC multidrug-resistance transporter that also mediates cell fusion, stem cell function, and vasculogenic plasticity. Immunohistochemistry and double-labeling immunofluorescence staining for ABCB5 and ABCB5/CD200, respectively, was performed on formalin-fixed, paraffin-embedded placental tissue from 5 first trimester, 5 second trimester, and 5 term pregnancies as well as 5 partial moles, and 5 complete moles. In addition, tumor cells from 5 choriocarcinoma and 5 placental site trophoblastic tumor cases were examined. ABCB5 staining was observed in villous trophoblasts in 100% (5/5) of first trimester placentas (with progressive decrease in term placentas); 100% of partial moles (5/5); and 100% of complete moles (5/5). Notably, reactivity was discretely restricted to the inner trophoblast layer, with no staining of overlying syncytiotrophoblast. Antibody specificity and localization was confirmed further by in situ hybridization. ABCB5 expression was retained in 20% of choriocarcinomas (1/5) and 40% of placental site trophoblastic tumors (2/5). Prior studies have localized expression of multidrug-resistance-1, also known as ABCB1, within the syncytiotrophoblast of early placentas, where it serves a protective function as an efflux transporter. Our results show that ABCB5 is preferentially expressed in the cytotrophoblast layer of placental villi. The expression of this novel biomarker at the maternal-fetal interface raises questions on its role in placental structure and function as well as on its potential contribution to the protective efflux provided by other P-glycoprotein transporters.

  9. Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum

    PubMed Central

    2013-01-01

    Background The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. Results We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. Conclusions The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control. PMID:23324493

  10. 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

  11. Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters.

    PubMed

    Leslie, E M; Deeley, R G; Cole, S P

    2001-10-05

    The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.

  12. 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

  13. 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....

  14. 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.

  15. Divide and conquer: processive transport enables multidrug transporters to tackle challenging drugs

    PubMed Central

    Fluman, Nir; Bibi, Eitan

    2014-01-01

    Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner. PMID:28357213

  16. 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.

  17. Function and frustration of multi-drug ABC exporter protein and design of model proteins for drug delivery using protein hydration thermodynamics.

    PubMed

    Urry, Dan W; Urry, Kelley D; Szaflarski, Witold; Nowicki, Michal; Zabel, Maciej

    2009-01-01

    The mechanism is presented whereby simultaneous hydrolysis of two molecules of ATP in the ATP-binding cassette (ABC) exporter protein, Sav 1866, opens a transmembrane channel to pump drug out of the cell and confers drug resistance, e.g., gives rise to methicillin resistant Staphylococcus aureus, MRSA. The proposed mechanism suggests pharmaceutical design strategies for overloading the capacity of two molecules of ATP to open access to the channel for export. Structural homology of Staphylococcus aureus, Sav 1866, to human P-glyco-protein and MRP2, suggests a similar mechanism could be relevant to human carcinoma cells. The transport mechanism utilizes two thermodynamic quantities -DeltaG(HA), the change in Gibbs free energy for hydrophobic association, and DeltaG(ap), an apolar-polar repulsive free energy for hydration, derived from studies on designed elastic-contractile model proteins (ECMPs). These quantities also allow design of remarkably biocompatible ECMPs as drug delivery vehicles with remarkable control of release profiles and of ECMPs that provide the means of developing pharmaceuticals for blocking multi-drug resistance.

  18. Binding and inhibition of drug transport proteins by heparin: a potential drug transporter modulator capable of reducing multidrug resistance in human cancer cells.

    PubMed

    Chen, Yunliang; Scully, Michael; Petralia, Gloria; Kakkar, Ajay

    2014-01-01

    A major problem in cancer treatment is the development of resistance to chemotherapeutic agents, multidrug resistance (MDR), associated with increased activity of transmembrane drug transporter proteins which impair cytotoxic treatment by rapidly removing the drugs from the targeted cells. Previously, it has been shown that heparin treatment of cancer patients undergoing chemotherapy increases survival. In order to determine whether heparin is capable reducing MDR and increasing the potency of chemotherapeutic drugs, the cytoxicity of a number of agents toward four cancer cell lines (a human enriched breast cancer stem cell line, two human breast cancer cell lines, MCF-7 and MDA-MB-231, and a human lung cancer cell line A549) was tested in the presence or absence of heparin. Results demonstrated that heparin increased the cytotoxicity of a range of chemotherapeutic agents. This effect was associated with the ability of heparin to bind to several of the drug transport proteins of the ABC and non ABC transporter systems. Among the ABC system, heparin treatment caused significant inhibition of the ATPase activity of ABCG2 and ABCC1, and of the efflux function observed as enhanced intracellular accumulation of specific substrates. Doxorubicin cytoxicity, which was enhanced by heparin treatment of MCF-7 cells, was found to be under the control of one of the major non-ABC transporter proteins, lung resistance protein (LRP). LRP was also shown to be a heparin-binding protein. These findings indicate that heparin has a potential role in the clinic as a drug transporter modulator to reduce multidrug resistance in cancer patients.

  19. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  20. Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum▿

    PubMed Central

    Nataf, Yakir; Yaron, Sima; Stahl, Frank; Lamed, Raphael; Bayer, Edward A.; Scheper, Thomas-Helmut; Sonenshein, Abraham L.; Shoham, Yuval

    2009-01-01

    Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G2 to G5), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G3), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G3 to G5) are assimilated faster than cellobiose. PMID:18952792

  1. 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

  2. Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

    PubMed

    Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

    2017-02-01

    The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families.

  3. Lipids modulate the conformational dynamics of a secondary multidrug transporter

    PubMed Central

    Martens, Chloé; Stein, Richard A; Masureel, Matthieu; Roth, Aurélie; Mishra, Smriti; Dawaliby, Rosie; Konijnenberg, Albert; Sobott, Frank; Govaerts, Cédric; Mchaourab, Hassane S

    2017-01-01

    Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered, multidrug transporter LmrP from Lactococcus lactis utilizing the pattern of distances between spin label pairs previously shown to fingerprint alternating access of the protein. We uncover at the molecular level how the lipid headgroups shape the conformational energy landscape of the transporter. The model emerging from our data hypothesizes a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers. PMID:27399258

  4. 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

  5. Dual Repression of the Multidrug Efflux Pump CmeABC by CosR and CmeR in Campylobacter jejuni

    PubMed Central

    Grinnage-Pulley, Tara; Mu, Yang; Dai, Lei; Zhang, Qijing

    2016-01-01

    During transmission and intestinal colonization, Campylobacter jejuni, a major foodborne human pathogen, experiences oxidative stress. CosR, a response regulator in C. jejuni, modulates the oxidative stress response and represses expression of the CmeABC multidrug efflux pump. CmeABC, a key component in resistance to toxic compounds including antimicrobials and bile salts, is also under negative regulation by CmeR, a TetR family transcriptional regulator. How CosR and CmeR interact in binding to the cmeABC promoter and how CosR senses oxidative stress are still unknown. To answer these questions, we conducted various experiments utilizing electrophoretic mobility shift assays and transcriptional fusion assays. CosR and CmeR bound independently to two separate sites of the cmeABC promoter, simultaneously repressing cmeABC expression. This dual binding of CosR and CmeR is optimal with a 17 base pair space between the two binding sites as mutations that shortened the distance between the binding sites decreased binding by CmeR and enhanced cmeABC expression. Additionally, the single cysteine residue (C218) of CosR was sensitive to oxidation, which altered the DNA-binding activity of CosR and dissociated CosR from the cmeABC promoter as determined by electrophoretic mobility shift assay. Replacement of C218 with serine rendered CosR insensitive to oxidation, suggesting a potential role of C218 in sensing oxidative stress and providing a possible mechanism for CosR-mediated response to oxidative stress. These findings reveal a dual regulatory role of CosR and CmeR in modulating cmeABC expression and suggest a potential mechanism that may explain overexpression of cmeABC in response to oxidative stress. Differential expression of cmeABC mediated by CmeR and CosR in response to different signals may facilitate adaptation of Campylobacter to various environmental conditions. PMID:27468281

  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.

  7. Implicating ABC Transporters in Insecticide Resistance: Research Strategies and a Decision Framework.

    PubMed

    Gott, Ryan C; Kunkel, Grace R; Zobel, Emily S; Lovett, Brian R; Hawthorne, David J

    2017-02-28

    Pest insects damage crops, transmit diseases, and are household nuisances. Historically, they have been controlled with insecticides, but overuse often leads to resistance to one or more of these chemicals. Insects gain resistance to insecticides through behavioral, metabolic, genetic, and physical mechanisms. One frequently overlooked strategy is through the use of ATP-binding cassette (ABC) transporters. ABC transporters, present in all domains of life, perform natural excretory functions, thus the exploitation of these transporters to excrete insecticides and contribute to resistance is highly plausible. Previous work has implicated ABC transporters in some cases of insecticide resistance. Proposed herein is a framework meant as a formal guide for more easily incorporating the analysis of ABC transporters into existing resistance monitoring using suggested simple research methods. This framework functions as a simple decision tree and its utility is demonstrated using case examples. Determining a role for ABC transporters in insecticide resistance would help to shape future resistance management plans and guide the design of new insecticides.

  8. The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology.

    PubMed

    Cervenak, Judit; Andrikovics, Hajnalka; Ozvegy-Laczka, Csilla; Tordai, Attila; Német, Katalin; Váradi, András; Sarkadi, Balázs

    2006-03-08

    The human multidrug resistance ABC transporters provide a protective function in our body against a large number of toxic compounds. These proteins, residing in the plasma membrane, perform an active, ATP-dependent extrusion of such xenobiotics. However, the same proteins are also used by the tumor cells to fight various anticancer agents. ABCG2 is an important member of the multidrug resistance proteins, an 'ABC half transporter', which functions as a homodimer in the cell membrane. In this review, we provide a basic overview of ABCG2 function in physiology and drug metabolism, but concentrate on the discussion of mutations and polymorphisms discovered in this protein. Interestingly, a single nucleotide mutation, changing amino acid 482 from arginine to threonine or glycine in ABCG2, results in a major increase in the catalytic activity and a wider drug recognition by this protein. Still, this mutation proved to be an in vitro artifact, produced only in heavily drug-selected cell lines. In contrast, at least two, but possibly more polymorphic variants of ABCG2 were found to be present in large human populations with different ethnic background. However, currently available experimental data regarding the cellular expression, localization and function of these ABCG2 variants are strongly contradictory. Since, the proteins produced by these variant alleles may differently modulate cancer treatment, general drug absorption and toxicity, may represent risk factors in fetal toxicity, or alter the differentiation of stem cells, their exact characterization is a major challenge in this field.

  9. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  10. A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae.

    PubMed

    Baylay, Alison J; Ivens, Alasdair; Piddock, Laura J V

    2015-01-01

    Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.

  11. Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake.

    PubMed

    Balakrishnan, Lekshmy; Venter, Henrietta; Shilling, Richard A; van Veen, Hendrik W

    2004-03-19

    The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.

  12. 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...

  13. The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox).

    PubMed

    Szakács, Gergely; Váradi, András; Ozvegy-Laczka, Csilla; Sarkadi, Balázs

    2008-05-01

    ATP binding cassette (ABC) drug transporters play an important role in cancer drug resistance, protection against xenobiotics, and in general in the passage of drugs through cellular and tissue barriers. This review explores how human ABC transporters modulate the pharmacological effects of various drugs, and how this predictable ADME-TOX modulation can be used during the process of drug discovery and development. We provide a description of the relevant human ABC drug transporters and review the models and assay systems that can be applied for the analysis of their expected drug interactions. The use of the in vitro, in vivo, in silico models, their combination, and the emerging clinical information are evaluated with respect to their potential application in early drug screening.

  14. 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

  15. The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

    PubMed Central

    El-Awady, Raafat; Saleh, Ekram; Hashim, Amna; Soliman, Nehal; Dallah, Alaa; Elrasheed, Azza; Elakraa, Ghada

    2017-01-01

    Over the years chemotherapy failure has been a vital research topic as researchers have been striving to discover reasons behind it. The extensive studies carried out on chemotherapeutic agents confirm that resistance to chemotherapy is a major reason for treatment failure. “Resistance to chemotherapy,” however, is a comprehensive phrase that refers to a variety of different mechanisms in which ATP-binding cassette (ABC) mediated efflux dominates. The ABC is one of the largest gene superfamily of transporters among both eukaryotes and prokaryotes; it represents a variety of genes that code for proteins, which perform countless functions, including drug efflux – a natural process that protects cells from foreign chemicals. Up to date, chemotherapy failure due to ABC drug efflux is an active research topic that continuously provides further evidence on multiple drug resistance (MDR), aiding scientists in tackling and overcoming this issue. This review focuses on drug resistance by ABC efflux transporters in human, viral, parasitic, fungal and bacterial cells and highlights the importance of the MDR permeability glycoprotein being the mutual ABC transporter among all studied organisms. Current developments and future directions to overcome this problem are also discussed. PMID:28119610

  16. 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

  17. Effect of costunolide and dehydrocostus lactone on cell cycle, apoptosis, and ABC transporter expression in human soft tissue sarcoma cells.

    PubMed

    Kretschmer, Nadine; Rinner, Beate; Stuendl, Nicole; Kaltenegger, Heike; Wolf, Elisabeth; Kunert, Olaf; Boechzelt, Herbert; Leithner, Andreas; Bauer, Rudolf; Lohberger, Birgit

    2012-11-01

    Human soft tissue sarcomas represent a rare group of malignant tumours that frequently exhibit chemotherapeutic resistance and increased metastatic potential following unsuccessful treatment. In this study, we investigated the effects of costunolide and dehydrocostus lactone, which have been isolated from Saussurea lappa using activity-guided isolation, on three soft tissue sarcoma cell lines of various origins. The effects on cell proliferation, cell cycle distribution, apoptosis induction, and ABC transporter expression were analysed. Both compounds inhibited cell viability dose- and time-dependently. IC50 values ranged from 6.2 µg/mL to 9.8 µg/mL. Cells treated with costunolide showed no changes in cell cycle, little in caspase 3/7 activity, and low levels of cleaved caspase-3 after 24 and 48 h. Dehydrocostus lactone caused a significant reduction of cells in the G1 phase and an increase of cells in the S and G2/M phase. Moreover, it led to enhanced caspase 3/7 activity, cleaved caspase-3, and cleaved PARP indicating apoptosis induction. In addition, the influence of costunolide and dehydrocostus lactone on the expression of ATP binding cassette transporters related to multidrug resistance (ABCB1/MDR1, ABCC1/MRP1, and ABCG2/BCRP1) was examined using real-time RT-PCR. The expressions of ABCB1/MDR1 and ABCG2/BCRP1 in liposarcoma and synovial sarcoma cells were significantly downregulated by dehydrocostus lactone. Our data demonstrate for the first time that dehydrocostus lactone affects cell viability, cell cycle distribution and ABC transporter expression in soft tissue sarcoma cell lines. Furthermore, it led to caspase 3/7 activity as well as caspase-3 and PARP cleavage, which are indicators of apoptosis. Therefore, this compound may be a promising lead candidate for the development of therapeutic agents against drug-resistant tumours.

  18. 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

  19. Effect of Prostaglandin E2 on Multidrug Resistance Transporters In Human Placental Cells

    PubMed Central

    Lee, Gene T.; Dong, Yafeng; Zhou, Helen; He, Lily; Weiner, Carl P.

    2014-01-01

    Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades. PMID:25261564

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

    PubMed

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

    2015-10-01

    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.

  1. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and AdeRS mutations.

    PubMed

    Yoon, Eun-Jeong; Courvalin, Patrice; Grillot-Courvalin, Catherine

    2013-07-01

    Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system.

  2. Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

    PubMed

    Pérez-Boto, David; Acebo, Paloma; García-Peña, Francisco Javier; Abad, Juan Carlos; Echeita, María Aurora; Amblar, Mónica

    2015-06-01

    The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, β-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, β-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed β-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

  3. Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones.

    PubMed

    De Graef, J; Demeler, J; Skuce, P; Mitreva, M; Von Samson-Himmelstjerna, G; Vercruysse, J; Claerebout, E; Geldhof, P

    2013-04-01

    Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

  4. Regulation of ABC transporters blood-brain barrier: the good, the bad, and the ugly.

    PubMed

    Miller, David S

    2015-01-01

    The brain capillary endothelial cells that constitute the blood-brain barrier express multiple ABC transport proteins on the luminal, blood-facing, plasma membrane. These transporters function as ATP-driven efflux pumps for xenobiotics and endogenous metabolites. High expression of these ABC transporters at the barrier is a major obstacle to the delivery of therapeutics, including chemotherapeutics, to the CNS. Here, I review the signals that alter ABC transporter expression and transport function with an emphasis on P-glycoprotein, Mrp2, and breast cancer resistance protein (BCRP), the efflux transporters for which we have the most detailed picture of regulation. Recent work shows that transporter protein expression can be upregulated in response to inflammatory and oxidative stress, therapeutic drugs, diet, and persistent environmental pollutants; as a consequence, drug delivery to the brain is reduced (potentially bad and ugly). In contrast, basal transport activity of P-glycoprotein and BCRP can be reduced through complex signaling pathways that involve events in and on the brain capillary endothelial cells. Targeting these signaling events provides opportunities to rapidly and reversibly increase brain accumulation of drugs that are substrates for the transporters (potentially good). The clinical usefulness of targeting signaling to reduce efflux transporter activity and improve drug delivery to the CNS remains to be established.

  5. Heterocyclic cyclohexanone monocarbonyl analogs of curcumin can inhibit the activity of ATP-binding cassette transporters in cancer multidrug resistance.

    PubMed

    Revalde, Jezrael L; Li, Yan; Hawkins, Bill C; Rosengren, Rhonda J; Paxton, James W

    2015-02-01

    Curcumin (CUR) is a phytochemical that inhibits the xenobiotic ABC efflux transporters implicated in cancer multidrug resistance (MDR), such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins 1 and 5 (MRP1 and MRP5). The use of CUR in the clinic however, is complicated by its instability and poor pharmacokinetic profile. Monocarbonyl analogs of CUR (MACs) are compounds without CUR's unstable β-diketone moiety and were reported to have improved stability and in vivo disposition. Whether the MACs can be used as MDR reversal agents is less clear, as the absence of a β-diketone may negatively impact transporter inhibition. In this study, we investigated 23 heterocyclic cyclohexanone MACs for inhibitory effects against P-gp, BCRP, MRP1 and MRP5. Using flow cytometry and resistance reversal assays, we found that many of these compounds inhibited the transport activity of the ABC transporters investigated, often with much greater potency than CUR. Overall the analogs were most effective at inhibiting BCRP and we identified three compounds, A12 (2,6-bis((E)-2,5-dimethoxy-benzylidene)cyclohexanone), A13 (2,6-bis((E)-4-hydroxyl-3-methoxybenzylidene)-cyclohexanone) and B11 (3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one), as the most promising BCRP inhibitors. These compounds inhibited BCRP activity in a non-cell line, non-substrate-specific manner. Their inhibition occurred by direct transporter interaction rather than modulating protein or cell surface expression. From these results, we concluded that MACs, such as the heterocyclic cyclohexanone analogs in this study, also have potential as MDR reversal agents and may be superior alternatives to the unstable parent compound, CUR.

  6. The riboswitch regulates a thiamine pyrophosphate ABC transporter of the oral spirochete Treponema denticola.

    PubMed

    Bian, Jiang; Shen, Hongwu; Tu, Youbin; Yu, Aiming; Li, Chunhao

    2011-08-01

    Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B₁), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete Treponema denticola is an important pathogen that is associated with human periodontal diseases. It lacks a de novo TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (tbpABC(Td)) that is initiated by a σ⁷⁰-like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Td(thi-)(box)). Genetic and biochemical studies revealed that the TDE0143 deletion mutant (T. denticola ΔtbpA) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the tbpABC(Td) operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism.

  7. Multidrug resistance: a transport system of antitumor agents and xenobiotics.

    PubMed

    Tsuruo, T

    1990-01-01

    Resistance of tumors to a variety of chemotherapeutic agents presents a major problem in cancer treatment. Resistance to such agents as doxorubicin, Vinca alkaloids, and actinomycin D can be acquired by tumor cells after treatment with a single drug. The gene responsible for multidrug resistance, termed mdr1, encodes a membrane glycoprotein (P-glycoprotein) that acts as a pump to transport various cytotoxic agents including various xenobiotics out of the cell. The amount of P-glycoprotein expression has been measured in tumor samples and was found to be elevated in intrinsically drug-resistant cancers of the colon, kidney, and adrenal as well as in some tumors that acquired drug resistance after chemotherapy. The protein was also found to be elevated in cells treated with xenobiotics. P-glycoprotein has been shown to bind anticancer drugs and several resistance-reversing agents including calcium channel blockers, and to be an ATPase. We recently reconstituted the purified P-glycoprotein into artificial liposomes. Reconstituted P-glycoprotein showed ATPase activity, ATP-dependent drug-transport activity, and calcium channel blocker-binding activity. This model provides many advantages for studies of the biochemical functions of P-glycoprotein. In addition to these basic interests, the protein is of considerable interest as a target for cancer chemotherapy because it appears to be involved in both acquired multidrug resistance and intrinsic drug resistance in human cancer. The selective killing of tumor cells expressing P-glycoprotein could be very important in future cancer therapy.

  8. 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...

  9. The eefABC multidrug efflux pump operon is repressed by H-NS in Enterobacter aerogenes.

    PubMed

    Masi, Muriel; Pagès, Jean-Marie; Villard, Claude; Pradel, Elizabeth

    2005-06-01

    The Enterobacter aerogenes eefABC locus, which encodes a tripartite efflux pump, was cloned by complementation of an Escherichia coli tolC mutant. E. aerogenes deltaacrA expressing EefABC became less susceptible to a wide range of antibiotics. Data from eef::lacZ fusions showed that eefABC was not transcribed in the various laboratory conditions tested. However, increased transcription from Peef was observed in an E. coli hns mutant. In addition, EefA was detected in E. aerogenes expressing a dominant negative E. coli hns allele.

  10. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance.

    PubMed

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols.

  11. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance

    PubMed Central

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols. PMID:25254091

  12. Regulation of ABC Efflux Transporters at Blood-Brain Barrier in Health and Neurological Disorders

    PubMed Central

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

    2015-01-01

    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. PMID:26187753

  13. 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

  14. 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.

  15. Tyrosine kinase inhibitors as modulators of ABC transporter-mediated drug resistance

    PubMed Central

    Shukla, Suneet; Chen, Zhe-Sheng; Ambudkar, Suresh V.

    2012-01-01

    Tyrosine kinases (TKs) are involved in key signaling events/pathways that regulate cancer cell proliferation, apoptosis, angiogenesis and metastasis. Deregulated activity of TKs has been implicated in several types of cancers. In recent years, tyrosine kinase inhibitors (TKIs) have been developed to inhibit specific kinases whose constitutive activity results in specific cancer types. These TKIs have been found to demonstrate effective anticancer activity and some of them have been approved by the Food and Drug Administration for clinical use or are in clinical trials. However, these targeted therapeutic agents are also transported by ATP-binding cassette (ABC) transporters, resulting in altered pharmacokinetics or development of resistance to these drugs in cancer patients. This review covers the recent findings on the interactions of clinically important TKIs with ABC drug transporters. Future research efforts in the development of novel TKIs with specific targets, seeking improved activity, should consider these underlying causes of resistance to TKIs in cancer cells. PMID:22325423

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

    PubMed Central

    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. PMID:26696990

  17. 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.

  18. 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

  19. The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration

    PubMed Central

    Molday, Robert S.; Zhong, Ming; Quazi, Faraz

    2009-01-01

    ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders. PMID:19230850

  20. 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

  1. Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2.

    PubMed

    Bao, Huan; Duong, Franck

    2012-01-01

    The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.

  2. Transported Substrate Determines Exchange Rate in the Multidrug Resistance Transporter EmrE*

    PubMed Central

    Morrison, Emma A.; Henzler-Wildman, Katherine A.

    2014-01-01

    EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport. PMID:24448799

  3. Transported substrate determines exchange rate in the multidrug resistance transporter EmrE.

    PubMed

    Morrison, Emma A; Henzler-Wildman, Katherine A

    2014-03-07

    EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport.

  4. A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.

    PubMed

    Chang, Xiu-bao

    2007-03-01

    Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  5. 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.

  6. A Novel ATP-Binding Cassette Transporter Involved in Multidrug Resistance in the Phytopathogenic Fungus Penicillium digitatum

    PubMed Central

    Nakaune, Ryoji; Adachi, Kiichi; Nawata, Osamu; Tomiyama, Masamitsu; Akutsu, Katsumi; Hibi, Tadaaki

    1998-01-01

    Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Δpmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus. PMID:9758830

  7. 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

  8. Role of ABC transporters in fluoropyrimidine-based chemotherapy response.

    PubMed

    Nies, Anne T; Magdy, Tarek; Schwab, Matthias; Zanger, Ulrich M

    2015-01-01

    Since over 50 years, 5-fluorouracil (5-FU) is in use as backbone of chemotherapy treatment regimens for a wide range of cancers including colon, breast, and head and neck carcinomas. However, drug resistance and severe toxicities such as mucositis, diarrhea, neutropenia, and vomiting in up to 40% of treated patients often lead to dose limitation or treatment discontinuation. Because the oral bioavailability of 5-FU is unpredictable and highly variable, 5-FU is commonly administered intravenously. To overcome medical complications and inconvenience associated with intravenous administration, the oral prodrugs capecitabine and tegafur have been developed. Both fluoropyrimidines are metabolically converted intracellularly to 5-FU, which then needs metabolic activation to exert its damaging activity on RNA and DNA. The low response rates of 10-15% of 5-FU monotherapy can be improved by combination regimens of infusional 5-FU and leucovorin together with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI), thereby increasing response rates to 30-40%. The impact of metabolizing enzymes in the development of fluoropyrimidine toxicity and resistance has been studied in great detail. In addition, membrane drug transporters, which are critical determinants of intracellular drug concentrations, may play a role in occurrence of toxicity and development of resistance against fluoropyrimidine-based therapy as well. This review therefore summarizes current knowledge on the role of drug transporters with particular focus on ATP-binding cassette transporters in fluoropyrimidine-based chemotherapy response.

  9. Alzheimer's and ABC transporters--new opportunities for diagnostics and treatment.

    PubMed

    Pahnke, Jens; Langer, Oliver; Krohn, Markus

    2014-12-01

    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.

  10. ABC transporters in CSCs membranes as a novel target for treating tumor relapse

    PubMed Central

    Zinzi, Laura; Contino, Marialessandra; Cantore, Mariangela; Capparelli, Elena; Leopoldo, Marcello; Colabufo, Nicola A.

    2014-01-01

    CSCs are responsible for the high rate of recurrence and chemoresistance of different types of cancer. The current antineoplastic agents able to inhibit bulk replicating cancer cells and radiation treatment are not efficacious toward CSCs since this subpopulation has several intrinsic mechanisms of resistance. Among these mechanisms, the expression of ATP-Binding Cassette (ABC) transporters family and the activation of different signaling pathways (such as Wnt/β-catenin signaling, Hedgehog, Notch, Akt/PKB) are reported. Therefore, considering ABC transporters expression on CSCs membranes, compounds able to modulate MDR could induce cytotoxicity in these cells disclosing an exciting and alternative strategy for targeting CSCs in tumor therapy. The next challenge in the cure of cancer relapse may be a multimodal strategy, an approach where specific CSCs targeting drugs exert simultaneously the ability to circumvent tumor drug resistance (ABC transporters modulation) and cytotoxic activity toward CSCs and the corresponding differentiated tumor cells. The efficacy of suggested multimodal strategy could be probed by using several scaffolds active toward MDR pumps on CSCs isolated by tumor specimens. PMID:25071581

  11. Interaction of forskolin with the P-glycoprotein multidrug transporter

    SciTech Connect

    Ming s, D.I.; Seamon, K.B. ); Speicher, L.A.; Tew, K.D. ); Ruoho, A.E. )

    1991-08-27

    Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug reing ance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, and 6-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and aing lyl cyclase, respectively. Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR. The ability of forskolin photolabels to specifically label the transporter, the adenylyl cyclase, and the P-glycoprotein suggests that these proteins may share a common biing g domain for forskolin analogues.

  12. Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica

    PubMed Central

    Shitan, Nobukazu; Bazin, Ingrid; Dan, Kazuyuki; Obata, Kazuaki; Kigawa, Koji; Ueda, Kazumitsu; Sato, Fumihiko; Forestier, Cyrille; Yazaki, Kazufumi

    2003-01-01

    Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome. PMID:12524452

  13. Functional Characterization of Candida albicans ABC Transporter Cdr1p

    PubMed Central

    Shukla, Suneet; Saini, Preeti; Smriti; Jha, Sudhakar; Ambudkar, Suresh V.; Prasad, Rajendra

    2003-01-01

    In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s). PMID:14665469

  14. Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes.

    PubMed

    Vajrala, Neeraja; Sayavedra-Soto, Luis A; Bottomley, Peter J; Arp, Daniel J

    2010-11-01

    Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe(3+)-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe(3+)-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe(3+) bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe(3+) or Fe(2+) forms or Fe(3+) associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.

  15. The choreography of multidrug export.

    PubMed

    Doshi, Rupak; Gutmann, Daniel A P; Khoo, Yvonne S K; Fagg, Lisa A; van Veen, Hendrik W

    2011-06-01

    Multidrug transporters have a crucial role in causing the drug resistance that can arise in infectious micro-organisms and tumours. These integral membrane proteins mediate the export of a broad range of unrelated compounds from cells, including antibiotics and anticancer agents, thus reducing the concentration of these compounds to subtoxic levels in target cells. In spite of intensive research, it is not clear exactly how multidrug transporters work. The present review focuses on recent advancements in the biochemistry and structural biology of bacterial and human multidrug ABC (ATP-binding cassette) transporters. These advancements point to a common mechanism in which polyspecific drug-binding surfaces in the membrane domains are alternately exposed to the inside and outside surface of the membrane in response to the ATP-driven dimerization of nucleotide-binding domains and their dissociation following ATP hydrolysis.

  16. Multidrug resistance in fungi: regulation of transporter-encoding gene expression

    PubMed Central

    Paul, Sanjoy; Moye-Rowley, W. Scott

    2014-01-01

    A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought. PMID:24795641

  17. Structural Features of the ATP-Binding Cassette (ABC) Transporter ABCA3

    PubMed Central

    Paolini, Alessandro; Baldassarre, Antonella; Del Gaudio, Ilaria; Masotti, Andrea

    2015-01-01

    In this review we reported and discussed the structural features of the ATP-Binding Cassette (ABC) transporter ABCA3 and how the use of bioinformatics tools could help researchers to obtain a reliable structural model of this important transporter. In fact, a model of ABCA3 is still lacking and no crystallographic structures (of the transporter or of its orthologues) are available. With the advent of next generation sequencing, many disease-causing mutations have been discovered and many more will be found in the future. In the last few years, ABCA3 mutations have been reported to have important pediatric implications. Thus, clinicians need a reliable structure to locate relevant mutations of this transporter and make genotype/phenotype correlations of patients affected by ABCA3-related diseases. In conclusion, we strongly believe that the model preliminarily generated by these novel bioinformatics tools could be the starting point to obtain more refined models of the ABCA3 transporter. PMID:26295388

  18. Jump into a New Fold—A Homology Based Model for the ABCG2/BCRP Multidrug Transporter

    PubMed Central

    László, Laura; Sarkadi, Balázs

    2016-01-01

    ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout. PMID:27741279

  19. Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

    PubMed Central

    Egner, R; Mahé, Y; Pandjaitan, R; Kuchler, K

    1995-01-01

    Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole. PMID:7565740

  20. 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.

  1. An ABC Transporter Is Required for Secretion of Peptide Sex Pheromones in Enterococcus faecalis

    PubMed Central

    Varahan, Sriram; Harms, Nathan; Gilmore, Michael S.; Tomich, John M.

    2014-01-01

    ABSTRACT Enterococci are leading causes of hospital-acquired infection in the United States and continue to develop resistances to new antibiotics. Many Enterococcus faecalis isolates harbor pheromone-responsive plasmids that mediate horizontal transfer of even large blocks of chromosomal genes, resulting in hospital-adapted strains over a quarter of whose genomes consist of mobile elements. Pheromones to which the donor cells respond derive from lipoprotein signal peptides. Using a novel bacterial killing assay dependent on the presence of sex pheromones, we screened a transposon mutant library for functions that relate to the production and/or activity of the effector pheromone. Here we describe a previously uncharacterized, but well-conserved, ABC transporter that contributes to pheromone production. Using three distinct pheromone-dependent mating systems, we show that mutants defective in expressing this transporter display a 5- to 6-order-of-magnitude reduction in conjugation efficiency. In addition, we demonstrate that the ABC transporter mutant displays an altered biofilm architecture, with a significant reduction in biofilm biomass compared to that of its isogenic parent, suggesting that pheromone activity also influences biofilm development. The conservation of this peptide transporter across the Firmicutes suggests that it may also play an important role in cell-cell communication in other species within this important phylum. PMID:25249282

  2. 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

  3. Natural variation in an ABC transporter gene associated with seed size evolution in tomato species.

    PubMed

    Orsi, Cintia Hotta; Tanksley, Steven D

    2009-01-01

    Seed size is a key determinant of evolutionary fitness in plants and is a trait that often undergoes tremendous changes during crop domestication. Seed size is most often quantitatively inherited, and it has been shown that Sw4.1 is one of the most significant quantitative trait loci (QTLs) underlying the evolution of seed size in the genus Solanum-especially in species related to the cultivated tomato. Using a combination of genetic, developmental, molecular, and transgenic techniques, we have pinpointed the cause of the Sw4.1 QTL to a gene encoding an ABC transporter gene. This gene exerts its control on seed size, not through the maternal plant, but rather via gene expression in the developing zygote. Phenotypic effects of allelic variation at Sw4.1 are manifested early in seed development at stages corresponding to the rapid deposition of starch and lipids into the endospermic cells. Through synteny, we have identified the Arabidopsis Sw4.1 ortholog. Mutagenesis has revealed that this ortholog is associated with seed length variation and fatty acid deposition in seeds, raising the possibility that the ABC transporter may modulate seed size variation in other species. Transcription studies show that the ABC transporter gene is expressed not only in seeds, but also in other tissues (leaves and roots) and, thus, may perform functions in parts of the plants other than developing seeds. Cloning and characterization of the Sw4.1 QTL gives new insight into how plants change seed during evolution and may open future opportunities for modulating seed size in crop plants for human purposes.

  4. The multidrug transporters belonging to major facilitator superfamily in Mycobacterium tuberculosis.

    PubMed Central

    De Rossi, Edda; Arrigo, Patrizio; Bellinzoni, Marco; Silva, Pedro A. E.; Martín, Carlos; Aínsa, José A.; Guglierame, Paola; Riccardi, Giovanna

    2002-01-01

    BACKGROUND: Both intrinsic and acquired multidrug resistance play an important role in the insurgence of tuberculosis. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors that block the multidrug transporter and allow traditional antibiotics to be effective. MATERIALS AND METHODS: We have undertaken the inventory of the drug transporters subfamily, included in the major facilitator superfamily (MFS), encoded by the complete genome of Mycobacterium tuberculosis (MTB). These proteins were identified on the basis of their characteristic stretches of amino acids and transmembrane segments (TMS) number. CONCLUSIONS: Genome analysis and searches of homology between the identified transporters and proteins characterized in other organisms revealed 16 open reading frames encoding putative drug efflux pumps belonging to MFS. In the case of two of them, we also have demonstrated that they function as drug efflux proteins. PMID:12520088

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

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

    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

  6. 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.

  7. 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

  8. Insight into determinants of substrate binding and transport in a multidrug efflux protein.

    PubMed

    Alegre, Kamela O; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J

    2016-03-10

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.

  9. Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P‐Glycoprotein (P‐gp) and Breast Cancer Resistance Protein (BCRP)

    PubMed Central

    Schwarz, Theresa; Montanari, Floriane; Cseke, Anna; Wlcek, Katrin; Visvader, Lene; Palme, Sarah; Chiba, Peter; Kuchler, Karl; Urban, Ernst

    2016-01-01

    Abstract The transmembrane ABC transporters P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug–drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand‐transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P‐gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC‐transporters. PMID:26970257

  10. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

    PubMed Central

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

  11. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters.

    PubMed

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.

  12. A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

    PubMed Central

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-01-01

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

  13. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

    PubMed

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-02-20

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

  14. Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

    PubMed Central

    Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

    2015-01-01

    Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters. PMID:26238402

  15. 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

  16. Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression.

    PubMed

    Fonseca, Elza; Silva, Sónia; Rodrigues, Célia Fortuna; Alves, Carlos Tiago; Azeredo, Joana; Henriques, Mariana

    2014-01-01

    Candida glabrata has emerged as the second most prevalent fungal pathogen and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms present a high tolerance to antifungal agents used in fungal infection treatment. The mechanisms of biofilm tolerance to antifungal agents remain poorly understood. Thus, the aim of this study was to evaluate the effects of fluconazole (FLU) on the formation and control of C. glabrata biofilms and its relation with the expression of genes encoding for ABC transporters, CDR1, SNQ2, and PDR1. For that, minimal inhibitory concentration values for seven C. glabrata strains were determined and the effect of FLU against C. glabrata biofilms was evaluated by total biomass quantification and viable cell enumeration. Matrices from biofilms were analyzed in terms of protein, carbohydrate and DNA content. ABC transporter gene expression was analyzed for quantitative real-time PCR. In addition to the high amounts of proteins and carbohydrates detected in the extracellular matrices in the presence of FLU, this work showed that the overexpression of efflux pumps is a possible mechanism of biofilm tolerance to FLU and this phenomenon alters the structure of C. glabrata biofilms by creating cell clusters.

  17. Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants.

    PubMed

    Mentewab, Ayalew; Stewart, C Neal

    2005-09-01

    Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.

  18. Rethinking Drug Treatment Approaches in ALS by Targeting ABC Efflux Transporters

    DTIC Science & Technology

    2012-10-01

    disease  progression,  and  found  that   chronic  treatment  with  either   the...down   disease  progression  as  reported.     In  addition  to  evaluating  the  toxicity  of   chronic  Elacridar...far underestimated issue of disease -driven pharmacoresistance mediated by the multi-drug resistance (mdr) efflux transporter, P-glycoprotein

  19. [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.

  20. Inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells.

    PubMed

    Lavie, Y; Harel-Orbital, T; Gaffield, W; Liscovitch, M

    2001-01-01

    Intrinsic or acquired resistance of tumor cells to multiple cytotoxic drugs (multidrug resistance MDR) is a major cause of failure of cancer chemotherapy. MDR is often caused by elevated expression of drug transporters such as P-glycoprotein (P-gp) or multidrug resistance protein (MRP). A number of compounds, termed chemosensitizers, have little or no cytotoxic action of their own, but inhibit (P-gp) or MRP-mediated drug export and are capable of sensitizing MDR cells to the cytotoxic effects of chemotherapeutic drugs. Here we examined the ability of steroidal alkaloids of plant origin, namely the Veratrum sp. alkaloid cyclopamine and the Lycopersicon sp. alkaloid tomatidine, to act as potent and effective chemosensitizers in multidrug resistant tumor cells. Drug uptake was determined by measuring accumulation of tetramethylrosamine in multidrug resistant NCI AdrR human adenocarcinoma cells. Resistance to adriamycin and vinblastine was determined by utilizing the MTT cell survival assay. Cyclopamine and tomatidine elevate tetramethylrosamine uptake by NCI AdrR cells and sensitize the cells to the cytotoxic action of adriamycin and vinblastine. In both cases these agents are comparable in patency and efficacy to verapamil, a reversal agent commonly used in MDR research. It is concluded that steroidal alkaloids of plant origin act as inhibitors of P-gp-mediated drug transport and multidrug resistance and therefore may serve as chemosensitizers in combination chemotherapy with conventional cytotoxic drugs for treating multidrug resistant cancer.

  1. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.

    PubMed

    Lu, Min; Symersky, Jindrich; Radchenko, Martha; Koide, Akiko; Guo, Yi; Nie, Rongxin; Koide, Shohei

    2013-02-05

    Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

  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. Cerebral ABC transporter-common mechanisms may modulate neurodegenerative diseases and depression in elderly subjects.

    PubMed

    Pahnke, Jens; Fröhlich, Christina; Paarmann, Kristin; Krohn, Markus; Bogdanovic, Nenad; Årsland, Dag; Winblad, Bengt

    2014-11-01

    In elderly subjects, depression and dementia often coincide but the actual reason is currently unknown. Does a causal link exist or is it just a reactive effect of the knowledge to suffer from dementia? The ABC transporter superfamily may represent a causal link between these mental disorders. Since the transporters ABCB1 and ABCC1 have been discovered as major β-amyloid-exporting molecules at the blood-brain barrier and ABCC1 was found to be directly activated by St. John's wort (SJW), depression and dementia certainly share an important pathophysiologic link. It was recognized that herbal anti-depressant formulations made from SJW are at least as effective for the treatment of unipolar depression in old age as classical pharmacotherapy, while having fewer side effects (Cochrane reports, 2008). SJW is known to activate various metabolizing and transport systems in the body, with cytochrome P450 enzymes and ABC transporters being most important. Does the treatment of depression in elderly subjects using pharmacological compounds or phytomedical extracts target a mechanism that also accounts for peptide storage in Alzheimer's disease and perhaps other proteopathies of the brain? In this review we summarize recent data that point to a common mechanism and present the first promising causal treatment results of demented elderly subjects with distinct SJW extracts. Insufficient trans-barrier clearance may indeed present a common problem in all the proteopathies of the brain where toxic peptides are deposited in a location-specific manner. Thus, activation of efflux molecules holds promise for future treatment of this large group of devastating disorders.

  4. A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

    PubMed Central

    Manzano, J. I.; García-Hernández, R.; Castanys, S.

    2013-01-01

    The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes. PMID:23716044

  5. 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

  6. 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

  7. A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species

    PubMed Central

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun

    2014-01-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

  8. Interactions of retinoids with the ABC transporters P-glycoprotein and Breast Cancer Resistance Protein

    PubMed Central

    Tarapcsák, Szabolcs; Szalóki, Gábor; Telbisz, Ágnes; Gyöngy, Zsuzsanna; Matúz, Krisztina; Csősz, Éva; Nagy, Péter; Holb, Imre J.; Rühl, Ralph; Nagy, László; Szabó, Gábor; Goda, Katalin

    2017-01-01

    Retinoids – derivatives of vitamin A – are important cell permeant signaling molecules that regulate gene expression through activation of nuclear receptors. P-glycoprotein (Pgp) and ABCG2 are plasma membrane efflux transporters affecting the tissue distribution of numerous structurally unrelated lipophilic compounds. In the present work we aimed to study the interaction of the above ABC transporters with retinoid derivatives. We have found that 13-cis-retinoic acid, retinol and retinyl-acetate inhibited the Pgp and ABCG2 mediated substrate transport as well as the substrate stimulated ATPase activity of these transporters. Interestingly, 9-cis-retinoic acid and ATRA (all-trans retinoic acid), both are stereoisomers of 13-cis-retinoic acid, did not have any effect on the transporters’ activity. Our fluorescence anisotropy measurements revealed that 13-cis-retinoic acid, retinol and retinyl-acetate selectively increase the viscosity and packing density of the membrane. Thus, the mixed-type inhibition of both transporters by retinol and ABCG2 by 13-cis-retinoic acid may be the collective result of direct interactions of these retinoids with the substrate binding site(s) and of indirect interactions mediated by their membrane rigidifying effects. PMID:28145501

  9. What do proton motive force driven multidrug resistance transporters have in common?

    PubMed

    Mazurkiewicz, Piotr; Driessen, Arnold J M; Konings, Wil N

    2005-01-01

    The extensive progress of genome sequencing projects in recent years has demonstrated that multidrug resistance (MDR) transporters are widely spread among all domains of life. This indicates that they play crucial roles in the survival of organisms. Moreover, antibiotic and chemotherapeutic treatments have revealed that microorganisms and cancer cells may use MDR transporters to fight the cytotoxic action of drugs. Currently, several MDR extrusion systems are being investigated in detail. It is expected that understanding of the molecular basis of multidrug recognition and the transport mechanisms will allow a more rational design of new drugs which either will not be recognized and expelled by or will efficiently inhibit the activity of the MDR transporters. MDR transporters either utilize ATP hydrolysis or an ion motive force as an energy source to drive drugs out of the cell. This review summarizes the recent progress in the field of bacterial proton motive force driven MDR transporters.

  10. Transport of lipophilic carboxylates is mediated by transmembrane helix 2 in multidrug transporter AcrB

    PubMed Central

    Oswald, Christine; Tam, Heng-Keat; Pos, Klaas M.

    2016-01-01

    The deployment of multidrug efflux pumps is a powerful defence mechanism for Gram-negative bacterial cells when exposed to antimicrobial agents. The major multidrug efflux transport system in Escherichia coli, AcrAB–TolC, is a tripartite system using the proton-motive force as an energy source. The polyspecific substrate-binding module AcrB uses various pathways to sequester drugs from the periplasm and outer leaflet of the inner membrane. Here we report the asymmetric AcrB structure in complex with fusidic acid at a resolution of 2.5 Å and mutational analysis of the putative fusidic acid binding site at the transmembrane domain. A groove shaped by the interface between transmembrane helix 1 (TM1) and TM2 specifically binds fusidic acid and other lipophilic carboxylated drugs. We propose that these bound drugs are actively displaced by an upward movement of TM2 towards the AcrB periplasmic porter domain in response to protonation events in the transmembrane domain. PMID:27982032

  11. 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

  12. An ABC transporter controls export of a Drosophila germ cell attractant.

    PubMed

    Ricardo, Sara; Lehmann, Ruth

    2009-02-13

    Directed cell migration, which is critical for embryonic development, leukocyte trafficking, and cell metastasis, depends on chemoattraction. 3-hydroxy-3-methylglutaryl coenzyme A reductase regulates the production of an attractant for Drosophila germ cells that may itself be geranylated. Chemoattractants are commonly secreted through a classical, signal peptide-dependent pathway, but a geranyl-modified attractant would require an alternative pathway. In budding yeast, pheromones produced by a-cells are farnesylated and secreted in a signal peptide-independent manner, requiring the adenosine triphosphate-binding cassette (ABC) transporter Ste6p. Here we show that Drosophila germ cell migration uses a similar pathway, demonstrating that invertebrate germ cells, like yeast cells, are attracted to lipid-modified peptides. Components of this unconventional export pathway are highly conserved, suggesting that this pathway may control the production of similarly modified chemoattractants in organisms ranging from yeast to humans.

  13. Comparison of Cytotoxicity and Inhibition of Membrane ABC Transporters Induced by MWCNTs with Different Length and Functional Groups.

    PubMed

    Yu, Jing; Liu, Su; Wu, Bing; Shen, Zhuoyan; Cherr, Gary N; Zhang, Xu-Xiang; Li, Mei

    2016-04-05

    Experimental studies indicate that multiwalled carbon nanotubes (MWCNTs) have the potential to induce cytotoxicity. However, the reports are often inconsistent and even contradictory. Additionally, adverse effects of MWCNTs at low concentration are not well understood. In this study, we systemically compared adverse effects of six MWCNTs including pristine MWCNTs, hydroxyl-MWCNTs and carboxyl-MWCNTs of two different lengths (0.5-2 μm and 10-30 μm) on human hepatoma cell line HepG2. Results showed that MWCNTs induced cytotoxicity by increasing reactive oxygen species (ROS) generation and damaging cell function. Pristine short MWCNTs induced higher cytotoxicity than pristine long MWCNTs. Functionalization increased cytotoxicity of long MWCNTs, but reduced cytotoxicity of short MWCNTs. Further, our results indicated that the six MWCNTs, at nontoxic concentration, might not be environmentally safe as they inhibited ABC transporters' efflux capabilities. This inhibition was observed even at very low concentrations, which were 40-1000 times lower than their effective concentrations on cytotoxicity. The inhibition of ABC transporters significantly increased cytotoxicity of arsenic, a known substrate of ABC transporters, indicating a chemosensitizing effect of MWCNTs. Plasma membrane damage was likely the mechanism by which the six MWCNTs inhibited ABC transporter activity. This study provides insight into risk assessments of low levels of MWCNTs in the environment.

  14. 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...

  15. Reversers of the multidrug resistance transporter P-glycoprotein.

    PubMed

    Stein, Wilfred D

    2002-05-01

    Multidrug resistance can arise from the presence of the membrane-bound pump, P-glycoprotein, in a tumor. Major efforts have been made to develop inhibitors of this pump, and a number of promising blockers have reached late stages of clinical trials. The kinetics of the inhibition of P-glycoprotein is complex, with binding sites that can interact synergistically. Reversers of increased affinity and specificity could, in principle, be developed on the basis of these synergies, and offer some promise in cancer therapeutics.

  16. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA.

    PubMed

    Sakamoto, K; Margolles, A; van Veen, H W; Konings, W N

    2001-09-01

    Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

  17. Hop Resistance in the Beer Spoilage Bacterium Lactobacillus brevis Is Mediated by the ATP-Binding Cassette Multidrug Transporter HorA

    PubMed Central

    Sakamoto, Kanta; Margolles, Abelardo; van Veen, Hendrik W.; Konings, Wil N.

    2001-01-01

    Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters. PMID:11514522

  18. 4-Isoxazolyl-1,4-dihydropyridines exhibit binding at the multidrug-resistance transporter.

    PubMed

    Hulubei, Victoria; Meikrantz, Scott B; Quincy, David A; Houle, Tina; McKenna, John I; Rogers, Mark E; Steiger, Scott; Natale, N R

    2012-11-15

    The 4-isoxazolyl-dihydropyridines (IDHPs) exhibit inhibition of the multidrug-resistance transporter (MDR-1), and exhibit an SAR distinct from their activity at voltage gated calcium channels (VGCC). Among the four most active IDHPs, three were branched at C-5 of the isoxazole, including the most active analog, 1k.

  19. Switch-Loop Flexibility Affects Transport of Large Drugs by the Promiscuous AcrB Multidrug Efflux Transporter

    PubMed Central

    Cha, Hi-jea; Müller, Reinke T.

    2014-01-01

    Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å2 are less well transported than other substrates. PMID:24914123

  20. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

    PubMed

    Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

    2013-02-01

    Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

  1. Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

    PubMed

    Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin-Verneuil, Aurélie; Blancato, Víctor S; Repizo, Guillermo D; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef

    2017-05-01

    Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically

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

    PubMed Central

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

    2013-01-01

    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 PMID:24302568

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

    PubMed

    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.

  4. 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.

  5. 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.

  6. Molecular cloning and characterization of Crmdr1, a novel MDR-type ABC transporter gene from Catharanthus roseus.

    PubMed

    Jin, Hongbin; Liu, Donghui; Zuo, Kaijing; Gong, Yifu; Miao, Zhiqi; Chen, Yuhui; Ren, Weiwei; Sun, Xiaofen; Tang, Kexuan

    2007-08-01

    A novel gene encoding a MDR-like ABC transporter protein was cloned from Catharanthus roseus, a medicinal plant with more than 120 kinds of secondary metabolites, through rapid amplification of cDNA ends (RACE). This gene (named as Crmdr1; GenBank accession no.: DQ660356) had a total length of 4395 bp with an open reading frame of 3801 bp, and encoded a predicted polypeptide of 1266 amino acids with a molecular weight of 137.1 kDa. The CrMDR1 protein shared 59.8, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAD31576), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Crmdr1 was a low-copy gene. Expression pattern analysis revealed that Crmdr1 constitutively expressed in the root, stem and leaf, but with lower expression in leaf. The domains analysis showed that CrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that CrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

  7. Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.

    PubMed

    Chang, Xiu-bao

    2010-01-01

    Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  8. Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

    PubMed Central

    Srinivas, Ranga V.; Middlemas, David; Flynn, Pat; Fridland, Arnold

    1998-01-01

    The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)—saquinavir, ritonavir, nelfinavir, and indinavir—interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1+ CEM/VBL100 and MRP1+ CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy. PMID:9835508

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

    PubMed Central

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

    2016-01-01

    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

  10. Structural Dynamics of the Heterodimeric ABC Transporter TM287/288 Induced by ATP and Substrate Binding.

    PubMed

    Furuta, Tadaomi; Sato, Yukiko; Sakurai, Minoru

    2016-12-06

    TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outward-facing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD-NBD communication. Furthermore, in the apo state, α-helical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a "core-exposed" model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters.

  11. Co-Induction of a Glutathione-S-transferase, a Glutathione Transporter and an ABC Transporter in Maize by Xenobiotics

    PubMed Central

    Liu, Zhiqian; Song, Xiaoyu; Li, Xuefeng; Wang, Chengju

    2012-01-01

    Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by four herbicides (2,4-D, atrazine, metolachlor and primisulfuron) and a herbicide safener (dichlormid) on the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect on gene expression varies with both chemicals and genes. The expression of ZmGST27 and ZmMRP1 was up-regulated by all five compounds, whereas that of ZmGT1 was increased by atrazine, metolachlor, primisulfuron and dichlormid, but not by 2,4-D. For all chemicals, the inducing effect was first detected on ZmGST27. The finding that ZmGT1 is activated alongside ZmGST27 and ZmMRP1 suggests that glutathione transporters are an important component in the xenobiotic detoxification system of plants. PMID:22792398

  12. Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.

    PubMed

    Della Torre, Camilla; Zaja, Roko; Loncar, Jovica; Smital, Tvrtko; Focardi, Silvano; Corsi, Ilaria

    2012-06-25

    The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

  13. Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro.

    PubMed

    Hsiao, Sung-Han; Lu, Yu-Jen; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung; Wu, Chung-Pu

    2016-06-06

    The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, which results in low oral bioavailability, reduced distribution, and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration. We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1 without affecting the expression level of ABCB1, enhances drug-induced apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors.

  14. Multidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the half ATP-binding cassette transporter ABCG2.

    PubMed

    Han, B; Zhang, J-T

    2004-01-01

    ABCG2, also termed BCRP/MXR/ABCP, is a half ATP-binding cassette (ABC) transporter expressed on plasma membranes. ABCG2 was independently cloned from placenta as well as cell lines selected for resistance to mitoxantrone or anthracyclines. ABCG2 consists of a nucleotide-binding domain (NBD) at the amino terminus and a transmembrane domain (TMD) at the carboxyl terminus and it is postulated to form a homodimer to perform its biological functions. Over-expression of ABCG2 in cell lines confers resistance on a wide variety of anticancer drugs including mitoxantrone, daunorubicin, doxorubicin, topotecan and epirubicin. The expression of ABCG2 has been implicated in multidrug resistance (MDR) of acute myeloid leukemia and some solid tumors. In addition, ABCG2 can transport several fluorescent dyes or toxins. ABCG2 is found to be expressed in epithelial cells of intestine and colon, liver canaliculi, and renal tubules, where it serves to eliminate the plasma level of orally administered anticancer drugs as well as ingested toxins. ABCG2 is found to be highly expressed in placenta and the luminal surface of microvessel endothelium blood-brain barrier where it may play a role in limiting the penetration of drugs, such as topotecan from the maternal plasma into the fetus and from blood to brain. A variety of inhibitors for ABCG2 including GF120918 may prove useful for sensitizing cancer cells to chemotherapy or altering the distribution of orally administered drug substrates of ABCG2. Interestingly, ABCG2 is also expressed highly in hematopoietic stem cells. However, the function of ABCG2 in stem cells is currently unknown, although it may provide protection to stem cells from a variety of xenobiotics.

  15. FK520 interacts with the discrete intrahelical amino acids of multidrug transporter Cdr1 protein and acts as antagonist to selectively chemosensitize azole-resistant clinical isolates of Candida albicans.

    PubMed

    Nim, Shweta; Rawal, Manpreet K; Prasad, Rajendra

    2014-06-01

    FK520, a homolog of antifungal FK506, displays fungicidal synergism with azoles in Candida albicans and inhibits drug efflux mediated by ABC multidrug transporter. This study establishes the molecular basis of interaction of FK520 with Cdr1 protein, which is one of the major ABC multidrug transporters of C. albicans. For this, we have exploited an in-house library of Cdr1 protein consisting of 252 mutant variants where the entire primary structure of the two transmembrane domains comprising of 12 transmembrane helices was subjected to alanine scanning. With these mutant variants of Cdr1 protein, we could identify the critical amino acids of the transporter protein, which if replaced with alanine, not only abrogated FK520-dependent competitive inhibition of drug efflux but simultaneously decreased susceptibility to azoles. Notably, the replacement of most of the residues with alanine was inconsequential; however, there were close to 13% mutant variants, which showed abrogation of drug efflux and reversal of fungicidal synergy with azoles. Of note, all the intrahelical residues of Cdr1 protein, which abrogated inhibitor's ability to block the efflux and reversed fungicidal synergy, were common. Taken together, our results provide evidence of cross-talk of FK520 with Cdr1 by interacting with the select intrahelical residues of the protein to chemosensitize isolates of Candida.

  16. 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

  17. Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5.

    PubMed

    Urzúa, Lucia Soto; Vázquez-Candanedo, Ada P; Sánchez-Espíndola, Adriana; Ramírez, Carlos Ávila; Baca, Beatriz E

    2013-06-01

    Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.

  18. Alkyl-Lysophospholipid Resistance in Multidrug-Resistant Leishmania tropica and Chemosensitization by a Novel P-Glycoprotein-Like Transporter Modulator

    PubMed Central

    Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Parodi-Talice, Adriana; Jiménez, Ignacio A.; Ravelo, Angel G.; Castanys, Santiago; Gamarro, Francisco

    2001-01-01

    Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C5-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter. PMID:11502516

  19. 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

  20. H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB.

    PubMed

    Zhou, Yan; Ojeda-May, Pedro; Pu, Jingzhi

    2013-10-14

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters form a family of molecular motor proteins that couple ATP hydrolysis to substrate translocation across cell membranes. Each nucleotide binding domain of ABC-transporters contains a highly conserved H-loop histidine residue, whose precise mechanistic role in motor functions has remained elusive. By using combined quantum mechanical and molecular mechanical (QM/MM) calculations, we showed that the conserved H-loop residue H662 in E. coli HlyB, a bacterial ABC-transporter, can act first as a general acid and then as a general base to facilitate proton transfer in ATP hydrolysis. Without the assistance of H662, direct proton transfer from the lytic water to ATP results in a substantially higher barrier height. Our findings suggest that the essential function of the H-loop residue H662 is to provide a "chemical linchpin" that shuttles protons between reactants through a relay mechanism, thereby catalyzing ATP hydrolysis in HlyB.

  1. 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.

  2. 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

  3. 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

    2014-11-04

    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.

  4. The Signaling Interface of the Yeast Multidrug Transporter Pdr5 Adopts a Cis Conformation and There Is Functional Overlap and Equivalence of the Deviant and Canonical Q-loop Residues†

    PubMed Central

    Ananthaswamy, Neeti; Rutledge, Robert; Sauna, Zuben E.; Ambudkar, Suresh V.; Dine, Elliot; Nelson, Emily; Xia, Di; Golin, John

    2010-01-01

    ABC transporters are polytopic proteins. ATP hydrolysis and substrate transport take place in separate domains, and these activities must be coordinated through a signal interface. We previously characterized a mutation (S558Y) in the yeast multidrug transporter Pdr5 that uncouples ATP hydrolysis and drug transport. To characterize the transmission interface, we used a genetic screen to isolate second-site mutations of S558Y that restore drug transport. We recovered suppressors that restore drug resistance; their locations provide functional evidence for an interface in the cis rather than the trans configuration indicated by structural and crosslinking studies of bacterial and eukaryotic efflux transporters. One mutation, E244G, defines the Q-loop of the deviant portion of NBD1, which is the hallmark of this group of fungal transporters. When moved to an otherwise wild-type background, this mutation and its counterpart in the canonical ATP-binding site Q951G show a similar reduction in drug resistance and in the very high basal-level ATP hydrolysis characteristic of Pdr5. A double E244G, Q951G mutant is considerably more drug sensitive than either of the single mutations. Surprisingly, then, the deviant and canonical Q-loop residues are functionally overlapping and equivalent in a strikingly asymmetric ABC transporter. PMID:20426485

  5. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

    PubMed

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar; Haque, Abdul; De Zorzi, Rita; Mirza, Osman; Walz, Thomas; Rahman, Moazur

    2015-05-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

  6. Spin Labeling Studies of Transmembrane Signaling and Transport: Applications to Phototaxis, ABC Transporters and Symporters.

    PubMed

    Klare, Johann P; Steinhoff, Heinz-Jürgen

    2015-01-01

    Membrane proteins still represent a major challenge for structural biologists. This chapter will focus on the application of continuous wave and pulsed EPR spectroscopy on spin-labeled membrane proteins. Site-directed spin labeling EPR spectroscopy has evolved as a powerful tool to study the structure and dynamics of proteins, especially membrane proteins, as this method works largely independently of the size and complexity of the biological system under investigation. This chapter describes applications of this technique to three different systems: the archaeal photoreceptor/-transducer complex SRII/HtrII as an example for transmembrane signaling and two transport systems, the histidine ATP-binding cassette transporter HisQMP, and the sodium-proline symporter PutP.

  7. Unaltered expression of multidrug resistance transporters in polycyclic aromatic hydrocarbon-resistant rat liver cells.

    PubMed

    Payen, L; Courtois, A; Langouët, S; Guillouzo, A; Fardel, O

    2001-01-02

    Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2. F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine. They did not display either decreased cellular PAH accumulation or increased PAH efflux. In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity. Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells. These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells.

  8. Enantioselective induction of a glutathione-S-transferase, a glutathione transporter and an ABC transporter in maize by Metolachlor and its (S)-isomer.

    PubMed

    Pang, Sen; Ran, Zhaojin; Liu, Zhiqian; Song, Xiaoyu; Duan, Liusheng; Li, Xuefeng; Wang, Chengju

    2012-01-01

    The metabolism of chiral herbicides in plants remains poorly understood. Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by rac- and S-metolachlor of the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect of rac- and S-metolachlor on the expression of ZmGST27 and ZmGT1 is comparable. However, the inducing effect of rac-metolachlor on ZmMRP1 expression is more pronounced than that of S-metolachlor. Furthermore, vanadate, an ABC transporter inhibitor, could greatly reduce the difference in herbicidal activity between rac- and S-metolachlor. These results suggest that the ABC transporters may preferentially transport conjugates of rac-metolachlor, leading to a faster metabolism of the latter. Through comparing the expression of ZmGST27, ZmMRP1 and ZmGT1 after treatment by rac- and S-metolachlor, we provide novel insights into the metabolic processes of chiral herbicides in plants.

  9. Enantioselective Induction of a Glutathione-S-Transferase, a Glutathione Transporter and an ABC Transporter in Maize by Metolachlor and Its (S)-Isomer

    PubMed Central

    Liu, Zhiqian; Song, Xiaoyu; Duan, Liusheng; Li, Xuefeng; Wang, Chengju

    2012-01-01

    The metabolism of chiral herbicides in plants remains poorly understood. Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by rac- and S-metolachlor of the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect of rac- and S-metolachlor on the expression of ZmGST27 and ZmGT1 is comparable. However, the inducing effect of rac-metolachlor on ZmMRP1 expression is more pronounced than that of S-metolachlor. Furthermore, vanadate, an ABC transporter inhibitor, could greatly reduce the difference in herbicidal activity between rac- and S-metolachlor. These results suggest that the ABC transporters may preferentially transport conjugates of rac-metolachlor, leading to a faster metabolism of the latter. Through comparing the expression of ZmGST27, ZmMRP1 and ZmGT1 after treatment by rac- and S-metolachlor, we provide novel insights into the metabolic processes of chiral herbicides in plants. PMID:23144728

  10. Whole-transcriptome survey of the putative ATP-binding cassette (ABC) transporter family genes in the latex-producing laticifers of Hevea brasiliensis.

    PubMed

    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.

  11. Autophagy and Transporter-Based Multi-Drug Resistance

    PubMed Central

    Kumar, Priyank; Zhang, Dong-Mei; Degenhardt, Kurt; Chen, Zhe-Sheng

    2012-01-01

    All the therapeutic strategies for treating cancers aim at killing the cancer cells via apoptosis (programmed cell death type I). Defective apoptosis endow tumor cells with survival. The cell can respond to such defects with autophagy. Autophagy is a cellular process by which cytoplasmic material is either degraded to maintain homeostasis or recycled for energy and nutrients in starvation. A plethora of evidence has shown that the role of autophagy in tumors is complex. A lot of effort is needed to underline the functional status of autophagy in tumor progression and treatment, and elucidate how to tweak autophagy to treat cancer. Furthermore, during the treatment of cancer, the limitation for the cure rate and survival is the phenomenon of multi drug resistance (MDR). The development of MDR is an intricate process that could be regulated by drug transporters, enzymes, anti-apoptotic genes or DNA repair mechanisms. Reports have shown that autophagy has a dual role in MDR. Furthermore, it has been reported that activation of a death pathway may overcome MDR, thus pointing the importance of other death pathways to regulate tumor cell progression and growth. Therefore, in this review we will discuss the role of autophagy in MDR tumors and a possible link amongst these phenomena. PMID:24710490

  12. Identification of a meningococcal L-glutamate ABC transporter operon essential for growth in low-sodium environments.

    PubMed

    Monaco, Caterina; Talà, Adelfia; Spinosa, Maria Rita; Progida, Cinzia; De Nitto, Eleanna; Gaballo, Antonio; Bruni, Carmelo B; Bucci, Cecilia; Alifano, Pietro

    2006-03-01

    GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in l-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the l-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

  13. Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli.

    PubMed

    Hayashi, Mikiro; Tabata, Kazuhiko; Yagasaki, Makoto; Yonetani, Yoshiyuki

    2010-03-01

    L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.

  14. Structure of a cation-bound multidrug and toxic compound extrusion transporter

    SciTech Connect

    He, Xiao; Szewczyk, Paul; Karyakin, Andrey; Evin, Mariah; Hong, Wen-Xu; Zhang, Qinghai; Chang, Geoffrey

    2010-10-26

    Transporter proteins from the MATE (multidrug and toxic compound extrusion) family are vital in metabolite transport in plants, directly affecting crop yields worldwide. MATE transporters also mediate multiple-drug resistance (MDR) in bacteria and mammals, modulating the efficacy of many pharmaceutical drugs used in the treatment of a variety of diseases. MATE transporters couple substrate transport to electrochemical gradients and are the only remaining class of MDR transporters whose structure has not been determined. Here we report the X-ray structure of the MATE transporter NorM from Vibrio cholerae determined to 3.65 {angstrom}, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known MDR transporter. We also report a cation-binding site in close proximity to residues previously deemed critical for transport. This conformation probably represents a stage of the transport cycle with high affinity for monovalent cations and low affinity for substrates.

  15. 1.55 A structure of the ectoine binding protein TeaA of the osmoregulated TRAP-transporter TeaABC from Halomonas elongata.

    PubMed

    Kuhlmann, Sonja I; Terwisscha van Scheltinga, Anke C; Bienert, Ralf; Kunte, Hans-Jörg; Ziegler, Christine

    2008-09-09

    TeaABC from the moderate halophilic bacterium Halomonas elongata belongs to the tripartite ATP-independent periplasmic transporters (TRAP-T), a family of secondary transporters functioning in conjunction with periplasmic substrate binding proteins. TeaABC facilitates the uptake of the compatible solutes ectoine and hydroxyectoine that are accumulated in the cytoplasm under hyperosmotic stress to protect the cell from dehydration. TeaABC is the only known TRAP-T activated by osmotic stress. Currently, our knowledge on the osmoregulated compatible solute transporter is limited to ABC transporters or conventional secondary transporters. Therefore, this study presents the first detailed analysis of the molecular mechanisms underlying substrate recognition of the substrate binding protein of an osmoregulated TRAP-T. In the present study we were able to demonstrate by isothermal titration calorimetry measurements that TeaA is a high-affinity ectoine binding protein ( K d = 0.19 microM) that also has a significant but somewhat lower affinity to hydroxyectoine ( K d = 3.8 microM). Furthermore, we present the structure of TeaA in complex with ectoine at a resolution of 1.55 A and hydroxyectoine at a resolution of 1.80 A. Analysis of the TeaA binding pocket and comparison of its structure to other compatible solute binding proteins from ABC transporters reveal common principles in compatible solute binding but also significant differences like the solvent-mediated specific binding of ectoine to TeaA.

  16. Prevalence of Genes of OXA-23 Carbapenemase and AdeABC Efflux Pump Associated with Multidrug Resistance of Acinetobacter baumannii Isolates in the ICU of a Comprehensive Hospital of Northwestern China.

    PubMed

    Jia, Wei; Li, Caiyun; Zhang, Haiyun; Li, Gang; Liu, Xiaoming; Wei, Jun

    2015-08-21

    The objective of this study was to explore the molecular epidemiology and the genetic support of clinical multidrug resistant (MDR) Acinetobacter baumannii (A. baumannii) isolates in an ICU ward of a comprehensive hospital. A total of 102 non-duplicate drug-resistant A. baumannii isolates were identified and 93 (91.1%) of them were MDR strains. Molecular analysis demonstrated that carbapenemase genes blaOXA-23 and blaOXA-51 were presented in all 93 MDR isolates (100%), but other carbapenemase genes, including blaOXA-24, blaOXA-58, blaIMP-1, blaIMP-4, blaSIM, and blaVIM genes were completely absent in all isolates. In addition, genes of AdeABC efflux system were detected in 88.2% (90/102) isolates. Interestingly, an addition to efflux pump inhibitor, reserpine could significantly enhance the susceptibility of MDR isolates to moxifloxacin, cefotaxime, and imipenem (p < 0.01). Clonal relationship analysis further grouped these clinical drug-resistant isolates into nine clusters, and the MDR strains were mainly in clusters A, B, C, and D, which include 16, 13, 25, and 15 isolates, respectively. This study demonstrated that clinical isolates carrying carbapenemase-encoding genes blaOXA-23 and AdeABC efflux pump genes are the main prevalent MDR A. baumannii, and the co-expression of oxacillinase and efflux pump proteins are thus considered to be the important reason for the prevalence of this organism in the ICU of this hospital.

  17. Identification of TogMNAB, an ABC transporter which mediates the uptake of pectic oligomers in Erwinia chrysanthemi 3937.

    PubMed

    Hugouvieux-Cotte-Pattat, N; Blot, N; Reverchon, S

    2001-09-01

    The bacterium Erwinia chrysanthemi, which causes soft rot disease on various plants, is able to use pectin as a carbon source for growth. Knowledge of the critical step in pectin catabolism which allows the entry of pectic oligomers into the cells is scarce. We report here the first example of a transport system involved in the uptake of pectic oligomers. The TogMNAB transporter of E. chrysanthemi is a member of the ATP-binding cassette (ABC) superfamily. TogM and TogN are homologous to the inner membrane components, TogA exhibits the signature of ABC ATPases and TogB shows similarity with periplasmic ligand-binding proteins. The TogMNAB transporter is a new member of the carbohydrate uptake transporter-1 family (CUT1, TC no. 3.1.1), which is specialized in the transport of complex sugars. The four genes, togM, togN, togA and togB, are apparently co-transcribed in a large operon which also includes the pectate lyase gene pelW. The transcription of the tog operon is induced in the presence of pectic derivatives and is affected by catabolite repression. It is controlled by the KdgR repressor and the CRP activator. The TogMNAB system is able to provide Escherichia coli with the ability to transport oligogalacturonides. In E. chrysanthemi, the TogMNAB system seems to play a major role in switching on the induction of pectin catabolism. TogB also acts as a specific receptor for chemotaxis towards oligogalacturonides. The decreased capacity of maceration of a togM mutant indicates the importance of transport and/or attraction of oligogalacturonides for E. chrysanthemi pathogenicity.

  18. 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

  19. 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

  20. Structure and function of the universal stress protein TeaD and its role in regulating the ectoine transporter TeaABC of Halomonas elongata DSM 2581(T).

    PubMed

    Schweikhard, Eva S; Kuhlmann, Sonja I; Kunte, Hans-Jörg; Grammann, Katrin; Ziegler, Christine M

    2010-03-16

    The halophilic bacterium Halomonas elongata takes up the compatible solute ectoine via the osmoregulated TRAP transporter TeaABC. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. A transcriptional regulation via DNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 A by molecular replacement. TeaD forms a dimer-dimer complex with one ATP molecule bound to each monomer, which has a Rossmann-like alpha/beta overall fold. Our results reveal an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. USP-encoding orfs, which are located adjacent to genes encoding for TeaABC homologues, could be identified in several other organisms, and their physiological role in balancing the internal cellular ectoine pool is discussed.

  1. Variation and evolution of the ABC transporter genes ABCB1, ABCC1, ABCG2, ABCG5 and ABCG8: implication for pharmacogenetics and disease.

    PubMed

    Silverton, Latoya; Dean, Michael; Moitra, Karobi

    2011-01-01

    The ATP-binding cassette (ABC) transporter genes are ubiquitous in the genomes of all vertebrates. Some of these transporters play a key role in xenobiotic defense and are endowed with the capacity to efflux harmful toxic substances. A major role in the evolution of the vertebrate ABC genes is played by gene duplication. Multiple gene duplication and deletion events have been identified in ABC genes, resulting in either gene birth or gene death indicating that the process of gene evolution is still ongoing in this group of transporters. Additionally, polymorphisms in these genes are linked to variations in expression, function, drug disposition and drug response. Single nucleotide polymorphisms in the ABC genes may be considered as markers of individual risk for adverse drug reactions or susceptibility to complex diseases as they can uniquely influence the quality and quantity of gene product. As the ABC genes continue to evolve, globalization will yield additional migration and racial admixtures that will have far reaching implications for the pharmacogenetics of this unique family of transporters in the context of human health.

  2. 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

  3. 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.

  4. 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

  5. The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana.

    PubMed

    Stefanato, Francesca L; Abou-Mansour, Eliane; Buchala, Antony; Kretschmer, Matthias; Mosbach, Andreas; Hahn, Matthias; Bochet, Christian G; Métraux, Jean-Pierre; Schoonbeek, Henk-jan

    2009-05-01

    Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.

  6. The two-component system BfrAB regulates expression of ABC transporters in Streptococcus gordonii and Streptococcus sanguinis

    PubMed Central

    Zhang, Yongshu; Whiteley, Marvin; Kreth, Jens; Lei, Yu; Khammanivong, Ali; Evavold, Jamie N.; Fan, Jingyuan; Herzberg, Mark C.

    2009-01-01

    The putative two-component system BfrAB is involved in Streptococcus gordonii biofilm development. Here, we provide evidence that BfrAB regulates the expression of bfrCD and bfrEFG, which encode two ABC transporters, and bfrH, which encodes a CAAX amino-terminal protease family protein. BfrC and BfrE are ATP-binding proteins and BfrD, BfrF and BfrG are homologous membrane- spanning polypeptides. Similarly, BfrABss, the BfrAB homologous system in S. sanguinis controls the expression of two bfrCD-homologous operons (bfrCDss and bfrXYss), a bfrH-homologous gene (bfrH1ss) and another CAAX amino- terminal protease family protein gene (bfrH2ss). Furthermore, we demonstrate that the purified BfrA DNA-binding domain from S. gordonii binds to the promoter regions of bfrCD, bfrEFG, bfrH, bfrCDss, bfrXYss, and bfrH1ss in vitro. Finally, we show that the BfrA DNA-binding domain recognizes a conserved DNA motif with a consensuses sequence of TTTCTTTAGAAATATTTTAGAATT. These data suggest, therefore, that S. gordonii BfrAB could control biofilm formation by regulating multiple ABC-transporter systems. PMID:19118357

  7. The maltose ABC transporter in Lactococcus lactis facilitates high-level sensitivity to the circular bacteriocin garvicin ML.

    PubMed

    Gabrielsen, Christina; Brede, Dag A; Hernández, Pablo E; Nes, Ingolf F; Diep, Dzung B

    2012-06-01

    We generated and characterized a series of spontaneous mutants of Lactococcus lactis IL1403 with average 6- to 11-fold-lowered sensitivities to the circular bacteriocin garvicin ML (GarML). Carbohydrate fermentation assays highlighted changes in carbohydrate metabolism, specifically loss of the ability to metabolize starch and maltose, in these mutants. PCR and sequencing showed that a 13.5-kb chromosomal deletion encompassing 12 open reading frames, mainly involved in starch and maltose utilization, had spontaneously occurred in the GarML-resistant mutants. Growth experiments revealed a correlation between sensitivity to GarML and carbon catabolite repression (CCR); i.e., sensitivity to GarML increased significantly when wild-type cells were grown on maltose and galactose as sole carbohydrates, an effect which was alleviated by the presence of glucose. Among the genes deleted in the mutants were malEFG, which encode a CCR-regulated membrane-bound maltose ABC transporter. The complementation of mutants with these three genes recovered normal sensitivity to the bacteriocin, suggesting an essential role of the maltose ABC transporter in the antimicrobial activity of GarML. This notion was supported by the fact that the level of sensitivity to GarML was dose dependent, increasing with higher expression levels of malEFG over a 50-fold range. To our knowledge, this is the first time a specific protein complex has been demonstrated to be involved in sensitivity to a circular bacteriocin.

  8. 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

    2016-08-23

    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.

  9. Evidence for dual mode of action of a thiosemicarbazone, NSC73306: A potent substrate of the multidrug resistance-linked ABCG2 transporter

    PubMed Central

    Wu, Chung-Pu; Shukla, Suneet; Calcagno, Anna Maria; Hall, Matthew D.; Gottesman, Michael M.; Ambudkar, Suresh V.

    2008-01-01

    Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette transporters, including ABCB1 (P-glycoprotein), ABCG2 and several ABCC family members (MRPs). We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein and it re-sensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4 and MRP5. Our findings demonstrated that NSC73306 is not more toxic to cells that overexpress these transporters compared to their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140–150 nM concentration required for 50% stimulation) and by inhibition of [125I]-Iodoarylazidoprazosin photolabeling (50% inhibition at 250–400 nM) of the substrate-binding site(s). Overall, NSC73306 appears to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4 or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein-overexpressing cells, and by acting as a potent modulator that re-sensitizes ABCG2-expressing cancer cells to chemotherapeutics. PMID:18089722

  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 and splicing of ABC and SLC transporters in the human blood-brain barrier measured with RNAseq.

    PubMed

    Suhy, Adam M; Webb, Amy; Papp, Audrey C; Geier, Ethan G; Sadee, Wolfgang

    2017-02-07

    The blood-brain barrier (BBB) expresses numerous membrane transporters that supply needed nutrients to the central nervous system (CNS), consisting mostly of solute carriers (SLC transporters), or remove unwanted substrates via extrusion pumps through the action of ATP binding cassette (ABC) transporters. Previous work has identified many BBB transporters using hybridization arrays or qRT-PCR, using targeted probes. Here we have performed next-generation sequencing of the transcriptome (RNAseq) extracted from cerebral cortex tissues and brain microvessel endothelial cells (BMEC) obtained from two donors. The same RNA samples had previously been measured for transporter expression using qRT-PCR (Geier et al., 2013), yielding similar expression levels for overlapping mRNAs (R=0.66, p<0.001). RNAseq confirms a number of transporters highly enriched in BMECs (e.g., ABCB1, ABCG2, SLCO2B1, and SLC47A1), but also detects novel BMEC transporters. Multiple splice isoforms detected by RNAseq are either robustly enriched or depleted in BMECs, indicating differential RNA processing in the BBB. The Complete RNAseq data are publically available (GSE94064).

  12. Expression, detergent solubilization, and purification of a membrane transporter, the MexB multidrug resistance protein.

    PubMed

    Bhatt, Forum H; Jeffery, Constance J

    2010-12-03

    Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP). The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC). In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

  13. Carbohydrate kinase (RhaK)-dependent ABC transport of rhamnose in Rhizobium leguminosarum demonstrates genetic separation of kinase and transport activities.

    PubMed

    Rivers, Damien; Oresnik, Ivan J

    2013-08-01

    In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. This has led to a working hypothesis that RhaK has two biochemical functions: phosphorylation of its substrate and affecting the activity of the rhamnose ABC transporter. To address this hypothesis, a linker-scanning random mutagenesis of rhaK was carried out. Thirty-nine linker-scanning mutations were generated and mapped. Alleles were then systematically tested for their ability to physiologically complement kinase and transport activity in a strain carrying an rhaK mutation. The rhaK alleles generated could be divided into three classes: mutations that did not affect either kinase or transport activity, mutations that eliminated both transport and kinase activity, and mutations that affected transport activity but not kinase activity. Two genes of the last class (rhaK72 and rhaK73) were found to have similar biochemical phenotypes but manifested different physiological phenotypes. Whereas rhaK72 conferred a slow-growth phenotype when used to complement rhaK mutants, the rhaK73 allele did not complement the inability to use rhamnose as a sole carbon source. To provide insight to how these insertional variants might be affecting rhamnose transport and catabolism, structural models of RhaK were generated based on the crystal structure of related sugar kinases. Structural modeling suggests that both rhaK72 and rhaK73 affect surface-exposed residues in two distinct regions that are found on one face of the protein, suggesting that this protein's face may play a role in protein-protein interaction that affects rhamnose transport.

  14. Protonation-dependent conformational dynamics of the multidrug transporter EmrE

    PubMed Central

    Dastvan, Reza; Mishra, Smriti; Meiler, Jens; Mchaourab, Hassane S.

    2016-01-01

    The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1–3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient. PMID:26787875

  15. 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

  16. 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.

  17. 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

  18. A 20(S)-protopanoxadiol derivative overcomes multi-drug resistance by antagonizing ATP-binding cassette subfamily B member 1 transporter function

    PubMed Central

    Chen, Wantao; Xu, Qin; Xiao, Meng; Hu, Lihong; Mao, Li; Wang, Xu

    2016-01-01

    In cancer cells, failure of chemotherapy is often caused by the ATP-binding cassette subfamily B member 1 (ABCB1), and few drugs have been successfully developed to overcome ABCB1-mediated multi-drug resistance (MDR). To suppress ABCB1 activity, we previously designed and synthesized a new series of derivatives based on 20(S)-protopanoxadiol (PPD). In the present study, we investigated the role of PPD derivatives in the function of ABC transporters. Non-toxic concentrations of the PPD derivative PPD12 sensitized ABCB1-overexpressing cells to their anti-cancer substrates better than either the parental PPD or inactive PPD11. PPD12 increased intracellular accumulation of adriamycin and rhodamine123 in resistant cancer cells. Although PPD12 did not suppress the expression of ABCB1 mRNA or protein, it stimulated the activity of ABCB1 ATPase. Because PPD12 is a competitive inhibitor, it was predicted to bind to the large hydrophobic cavity of homology-modeled human ABCB1. PPD12 also enhanced the efficacy of adriamycin against ABCB1-overexpressing KB/VCR xenografts in nude mice. In conclusion, PPD12 enhances the efficacy of substrate drugs in ABCB1-overexpressing cancer cells. These findings suggest that a combination therapy consisting of PPD12 with conventional chemotherapeutic agents may be an effective treatment for ABCB1-mediated MDR cancer patients. PMID:26824187

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

    SciTech Connect

    DeGorter, Marianne K.; Conseil, Gwenaelle; Deeley, Roger G.; Campbell, Robert L.; Cole, Susan P.C.

    2008-01-04

    Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr{sup 324} in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.

  20. Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae.

    PubMed Central

    Baranova, N N; Neyfakh, A A

    1997-01-01

    A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy. PMID:9174208

  1. Glaucarubinone sensitizes KB cells to paclitaxel by inhibiting ABC transporters via ROS-dependent and p53-mediated activation of apoptotic signaling pathways

    PubMed Central

    Karthikeyan, Subburayan; Hoti, Sugeerappa Laxmanappa; Nazeer, Yasin; Hegde, Harsha Vasudev

    2016-01-01

    Multidrug resistance (MDR) is considered to be the major contributor to failure of chemotherapy in oral squamous cell carcinoma (SCC). This study was aimed to explore the effects and mechanisms of glaucarubinone (GLU), one of the major quassinoids from Simarouba glauca DC, in potentiating cytotoxicity of paclitaxel (PTX), an anticancer drug in KB cells. Our data showed that the administration of GLU pre-treatment significantly enhanced PTX anti-proliferative effect in ABCB1 over-expressing KB cells. The Rh 123 drug efflux studies revealed that there was a significant transport function inhibition by GLU-PTX treatment. Interestingly, it was also found that this enhanced anticancer efficacy of GLU was associated with PTX-induced cell arrest in the G2/M phase of cell cycle. Further, the combined treatment of GLU-PTX had significant decrease in the expression levels of P-gp, MRPs, and BCRP in resistant KB cells at both mRNA and protein levels. Furthermore, the combination treatments showed significant reactive oxygen species (ROS) production, chromatin condensation and reduced mitochondrial membrane potential in resistant KB cells. The results from DNA fragmentation analysis also demonstrated the GLU induced apoptosis in KB cells and its synergy with PTX. Importantly, GLU and/or PTX triggered apoptosis through the activation of pro-apoptotic proteins such as p53, Bax, and caspase-9. Our findings demonstrated for the first time that GLU causes cell death in human oral cancer cells via the ROS-dependent suppression of MDR transporters and p53-mediated activation of the intrinsic mitochondrial pathway of apoptosis. Additionally, the present study also focussed on investigation of the protective effect of GLU and combination drugs in human normal blood lymphocytes. Normal blood lymphocytes assay indicated that GLU is able to induce selective toxicity in cancer cells and in silico molecular docking studies support the choice of GLU as ABC inhibitor to enhance PTX efficacy

  2. Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter.

    PubMed

    Liu, R; Sharom, F J

    1997-03-11

    One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as an efflux pump for a diverse range of natural products, chemotherapeutic drugs, and hydrophobic peptides. In the present study, fluorescence techniques were used to probe the nucleotide binding domains (NBD) of P-glycoprotein. The transporter was labeled at two conserved cysteine residues, one within each NBD, using the thiol-reactive fluor 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS), and collisional quenching was used to assess solvent accessibility of the bound probe. Acrylamide was a poor quencher, which suggests that MIANS is buried in a relatively inaccessible region of the protein. Iodide ion was a highly effective quencher, whereas Cs+ was not, demonstrating the presence of a positive charge in the region close to the ATP binding site. The fluorescent nucleotide derivative 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) was hydrolysed slowly by P-glycoprotein, with a V(max) approximately 20-fold lower than that for unmodified ATP, and a K(M) of 81 microM. TNP-ATP and TNP-ADP inhibited P-glycoprotein ATPase activity, indicating that they interact with the NBD, whereas TNP-AMP was a very poor inhibitor. When TNP-nucleotides bound to P-glycoprotein, their fluorescence intensity was enhanced in a concentration-dependent manner. Both TNP-ATP and TNP-ADP bound to P-glycoprotein with substantially higher affinity than ATP, with K(d) values of 43 and 36 microM, respectively. Addition of ATP led to only partial displacement of TNP-ATP. Resonance energy transfer was observed between cysteine-bound MIANS and TNP-ATP/ADP, which indicated that the two fluorescent groups are located close to each other within the catalytic site of P-glycoprotein.

  3. 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

  4. 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...

  5. Tissue and developmental expression of a gene from Hessian fly encoding an ABC-active-transporter protein during interactions with wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the transcriptional patterns of a putative white (w) gene encoding an ABC-transporter protein during development in Hessian fly, Mayetiola destructor. The deduced amino acid sequence for the Hessian fly white showed 77 to 74% similarities to white/ATP-binding-cassette proteins and 57 t...

  6. The expression of superoxide dismutase (SOD) and a putative ABC transporter permease is inversely correlated during biofilm formation in Listeria monocytogenes 4b G

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC_transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant delta ...

  7. 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.

  8. 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.

  9. Substrate-dependent dynamics of the multidrug efflux transporter AcrB of Escherichia coli.

    PubMed

    Yamamoto, Kentaro; Tamai, Rei; Yamazaki, Megumi; Inaba, Takehiko; Sowa, Yoshiyuki; Kawagishi, Ikuro

    2016-02-26

    The resistance-nodulation-cell division (RND)-type xenobiotic efflux system plays a major role in the multidrug resistance of gram-negative bacteria. The only constitutively expressed RND system of Escherichia coli consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC. The latter two components are shared with another RND-type transporter AcrD, whose expression is induced by environmental stimuli. Here, we demonstrate how RND-type ternary complexes, which span two membranes and the cell wall, form in vivo. Total internal reflection fluorescence (TIRF) microscopy revealed that most fluorescent foci formed by AcrB fused to green fluorescent protein (GFP) were stationary in the presence of TolC but showed lateral displacements when tolC was deleted. The fraction of stationary AcrB-GFP foci decreased with increasing levels of AcrD. We propose that the AcrB-containing complex becomes unstable upon the induction of AcrD, which presumably replaces AcrB, a process we call "transporter exchange." This instability is suppressed by AcrB-specific substrates, suggesting that the ternary complex is stabilised when it is in action. These results suggest that the assembly of the RND-type efflux system is dynamically regulated in response to external stimuli, shedding new light on the adaptive antibiotic resistance of bacteria.

  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. The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

    PubMed Central

    2005-01-01

    The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a ‘flippase’ for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD–GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD–GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD–GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD–lactosylceramide drastically reduced the rate of flipping compared with NBD–PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids. PMID:15799713

  12. 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.

  13. Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in sea urchin embryos.

    PubMed

    Bosnjak, Ivana; Uhlinger, Kevin R; Heim, Wesley; Smital, Tvrtko; Franekić-Colić, Jasna; Coale, Kenneth; Epel, David; Hamdoun, Amro

    2009-11-01

    Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

  14. Activity of the dietary flavonoid, apigenin, against multidrug-resistant tumor cells as determined by pharmacogenomics and molecular docking.

    PubMed

    Saeed, Mohamed; Kadioglu, Onat; Khalid, Hassan; Sugimoto, Yoshikazu; Efferth, Thomas

    2015-01-01

    Apigenin is a common dietary flavonoid with considerable cytotoxic activity in vitro and in vivo. Despite many mechanistic studies, less is known about resistance factors hampering apigenin's activity. We investigated the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5. Multidrug-resistant cells overexpressing these ABC transporters were not cross-resistant toward apigenin. Moreover, apigenin inhibited not only P-glycoprotein but also BCRP by increasing cellular uptake of doxorubicin and synergistic inhibition of cell viability in combination with doxorubicin or docetaxel in multidrug-resistant cells. To perform in silico molecular docking studies, we first generated homology models for human P-glycoprotein and ABCB5 based on the crystal structure of murine P-glycoprotein. Their nucleotide binding domains (NDBs) revealed the highest degrees of sequence homologies (89%-100%), indicating that ATP binding and cleavage is of crucial importance for ABC transporters. Molecular docking of apigenin bound to the NDBs of P-glycoprotein and ABCB5 in molecular docking studies. Hence, apigenin may compete with ATP for NDB-binding leading to energy depletion to fuel the transport of ABC transporter substrates. Furthermore, we performed COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression profiles of the National Cancer Institute tumor cell line panel. Microarray-based mRNA expressions of genes of diverse biological functions (signal transduction, transcriptional regulation, ubiquitination, autophagy, metabolic activity, xenobiotic detoxification and microtubule formation) significantly predicted responsiveness of tumor cells to apigenin. In conclusion, apigenin's activity is not hampered by classical mechanisms of multidrug resistance and the inhibition of ABC transporters by apigenin indicates that apigenin may overcome multidrug resistance in otherwise refractory tumors.

  15. The enriched fraction of Elephantopus scaber Triggers apoptosis and inhibits multi-drug resistance transporters in human epithelial cancer cells

    PubMed Central

    Beeran, Asmy Appadath; Maliyakkal, Naseer; Rao, Chamallamudi Mallikarjuna; Udupa, Nayanabhirama

    2015-01-01

    Background: Medicinal plants have played an important role in the development of clinically useful anticancer agents. Elephantopus scaber (Asteraceae) (ES) is widely used in Indian traditional system of medicine for the treatment of various ailments including cancer. Objective: To investigate anticancer effects of ES in human epithelial cancer cells. Materials and Methods: Cytotoxicity of ethanolic extract of ES (ES-ET) and its fractions, such as ES Petroleum ether fraction (ES-PET), ES Dichloromethane fraction (ES DCM), n Butyl alcohol fraction (ES-BT), and ES-Rest (ES-R) were assessed in human epithelial cancer cell lines using sulforhodamine B (SRB) assay. Acridine orange/ethidium bromide assay and Hoechst 33342 assays were used to gauge induction of apoptosis. Cell cycle analysis and micronuclei assay were used to assess cell cycle specific pharmacological effects and drug induced genotoxicty. Further, the ability of ES to inhibit multi drug resistant (MDR) transporters (ABC-B1 and ABC-G2) was determined by Rhodamine (Rho) and Mitoxantrone (MXR) efflux assays. Results: The enriched fraction of ES (ES DCM) possessed dose-dependent potent cytotoxicity in human epithelial cancer cells. Further, treatment of cancer cells (HeLa, A549, MCF-7, and Caco-2) with ES DCM showed hall mark properties of apoptosis (membrane blebbing, nuclear condensation etc.). Similarly, ES DCM caused enhanced sub G0 content and micronuclei formation indicating the induction of apoptosis and drug induced genotoxicity in cancer cells, respectively. Interestingly, ES DCM inhibited MDR transporters (ABC B1 and ABC G2) in cancer cells. Conclusion: The enriched fraction of ES imparted cytotoxic effects, triggered apoptosis, induced genotoxicity, and inhibited MDR transporters in human epithelial cancer cells. Thus, ES appears to be potential anticancer agent. PMID:25829763

  16. An Arabidopsis ABC Transporter Mediates Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Roots.

    PubMed

    Dong, Jinsong; Piñeros, Miguel A; Li, Xiaoxuan; Yang, Haibing; Liu, Yu; Murphy, Angus S; Kochian, Leon V; Liu, Dong

    2017-02-13

    The remodeling of root architecture is a major developmental response of plants to phosphate (Pi) deficiency and is thought to enhance a plant's ability to forage for the available Pi in topsoil. The underlying mechanism controlling this response, however, is poorly understood. In this study, we identified an Arabidopsis mutant, hps10 (hypersensitive to Pi starvation 10), which is morphologically normal under Pi sufficient condition but shows increased inhibition of primary root growth and enhanced production of lateral roots under Pi deficiency. hps10 is a previously identified allele (als3-3) of the ALUMINUM SENSITIVE3 (ALS3) gene, which is involved in plant tolerance to aluminum toxicity. Our results show that ALS3 and its interacting protein AtSTAR1 form an ABC transporter complex in the tonoplast. This protein complex mediates a highly electrogenic transport in Xenopus oocytes. Under Pi deficiency, als3 accumulates higher levels of Fe(3+) in its roots than the wild type does. In Arabidopsis, LPR1 (LOW PHOSPHATE ROOT1) and LPR2 encode ferroxidases, which when mutated, reduce Fe(3+) accumulation in roots and cause root growth to be insensitive to Pi deficiency. Here, we provide compelling evidence showing that ALS3 cooperates with LPR1/2 to regulate Pi deficiency-induced remodeling of root architecture by modulating Fe homeostasis in roots.

  17. ABC transporters, CYP1A and GSTα gene transcription patterns in developing stages of the Nile tilapia (Oreochromis niloticus).

    PubMed

    Costa, Joana; Reis-Henriques, Maria Armanda; Castro, L Filipe C; Ferreira, Marta

    2012-09-15

    In fish, some ABC transporters are implicated in a multixenobiotic resistance (MXR) mechanism to deal with the presence of xenobiotics, by effluxing them, or their metabolites, from inside the cells. These efflux transporters have been considered an integral part of cellular detoxification pathways, acting in coordination with phase I and II detoxification enzymes. However, the full characterization of this detoxification system is still incomplete, especially during the developmental stages of aquatic organisms, which are particularly sensitive periods to the presence of anthropogenic contamination. The goal of this study was to evaluate the mRNA expression dynamics of putatively important MXR proteins (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2a) and phase I (CYP1A) and II (GSTα) biotransformation enzymes, during the embryonic and larval developments of the specie Oreochromis niloticus (Nile tilapia). Our results showed that ABCB1b, ABCC1, CYP1A and GSTα transcripts are maternally transmitted. Transcripts for ABCB11, ABCC2 and ABCG2a were only detected after the pharyngula period, which precedes a highly sensitive stage in the embryonic development, the hatching. This study has shown, for the first time, very distinct expression patterns of genes encoding for proteins involved in protection mechanisms against pollutants during the development of Nile tilapia. Moreover, the temporal pattern of gene expression suggests that increased intrinsic protection levels are required at specific developmental stages.

  18. 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.

  19. 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

  20. Circumvention of multi-drug resistance of cancer cells by Chinese herbal medicines

    PubMed Central

    2010-01-01

    Multi-drug resistance (MDR) of cancer cells severely limits therapeutic outcomes. A proposed mechanism for MDR involves the efflux of anti-cancer drugs from cancer cells, primarily mediated by ATP-binding cassette (ABC) membrane transporters including P-glycoprotein. This article reviews the recent progress of using active ingredients, extracts and formulae from Chinese medicine (CM) in circumventing ABC transporters-mediated MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. More mechanistic studies are needed to fully establish the pharmacological effects of potential MDR reversing agents. PMID:20653978

  1. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  2. Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems

    PubMed Central

    Liu, Qi; Henderson, Peter J. F.

    2015-01-01

    ABSTRACT Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. PMID:25670776

  3. P-glycoprotein multidrug transporter in inflammatory bowel diseases: More questions than answers

    PubMed Central

    Cario, Elke

    2017-01-01

    The gastrointestinal barrier is constantly exposed to numerous environmental substrates that are foreign and potentially harmful. These xenobiotics can cause shifts in the intestinal microbiota composition, affect mucosal immune responses, disturb tissue integrity and impair regeneration. The multidrug transporter ABCB1/MDR1 p-glycoprotein (p-gp) plays a key role at the front line of host defence by efficiently protecting the gastrointestinal barrier from xenobiotic accumulation. This Editorial discusses how altered expression and function of ABCB1/MDR1 p-gp may contribute to the development and persistence of chronic intestinal inflammation in inflammatory bowel diseases (IBD). Recent evidence implies multiple interactions between intestinal microbiota, innate immunity and xenobiotic metabolism via p-gp. While decreased efflux activity may promote disease susceptibility and drug toxicity, increased efflux activity may confer resistance to therapeutic drugs in IBD. Mice deficient in MDR1A develop spontaneously chronic colitis, providing a highly valuable murine IBD model for the study of intestinal epithelial barrier function, immunoregulation, infectious co-triggers and novel therapeutic approaches. Possible associations of human ABCB1 gene polymorphisms with IBD susceptibility have been evaluated, but results are inconsistent. Future studies must focus on further elucidation of the pathophysiological relevance and immunological functions of p-gp and how its ambiguous effects could be therapeutically targeted in IBD. PMID:28321153

  4. Flavonoids from Eight Tropical Plant Species That Inhibit the Multidrug Resistance Transporter ABCG2

    PubMed Central

    Versiani, Muhammad Ali; Diyabalanage, Thushara; Ratnayake, Ranjala; Henrich, Curtis J.; Bates, Susan E.; McMahon, James B.; Gustafson, Kirk R.

    2013-01-01

    Overexpression of ABCG2, a membrane-bound multidrug transporter, can make tumor cells resistant to treatment with conventional chemotherapeutic agents. A high-throughput screening effort with the NCI repository of natural product extracts revealed that eight tropical plant extracts significantly inhibited the function of ABCG2. This activity was tracked throughout the extract fractionation process to a series of ABCG2 inhibitory flavonoids (1–13). Their structures were identified by a combination of NMR, mass spectrometry, and circular dichroism studies, and this resulted in the elucidation of (2S)-5,7,3′-trihydroxy-4′-methoxy-8-(3″-methylbut-2″-enyl)-flavonone (1), (2S)-5,7,3′,5′-tetrahydroxy-8-[3″,8″ -dimethylocta-2″(E),7″-dienyl]flavonone (3), and 5,7,3′-trihydroxy-3,5′-dimethoxy-2′-(3′-methylbut-2-enyl)flavone (12) as new compounds. PMID:21275386

  5. Statistical thermodynamics for functionally rotating mechanism of the multidrug efflux transporter AcrB.

    PubMed

    Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Kinoshita, Masahiro

    2015-02-26

    AcrB, a homotrimer, is the pivotal part of a multidrug efflux pump. A "functionally rotating" picture has been proposed for the drug transport by AcrB, but its mechanism remains unresolved. Here, we investigate the energetics of the whole functional rotation cycle using our theoretical methods. We find that the packing efficiency of AcrB is ununiform, and this ununiformity plays imperative roles primarily through the solvent-entropy effect. When a proton binds to or dissociates from a protomer, the packing properties of this protomer and its two interfaces are perturbed overall in the direction that the solvent translational entropy is lowered. The packing properties of the other two protomers are then reorganized with the recovery or maintenance of closely packed interfaces, so that the solvent-entropy loss can be compensated. The functional structural change by an isolated protomer would cause a seriously large free-energy increase. By forming a trimer, any free-energy increase caused by a protomer is always canceled out by the free-energy decrease brought by the other two protomers via the mechanism mentioned above. The functional structural rotation is thus accomplished using the free-energy decrease arising from the transfer of only a single proton per cycle. The similarities to F1-ATPase are also discussed.

  6. Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization

    PubMed Central

    Galián, Carmen; Manon, Florence; Dezi, Manuela; Torres, Cristina; Ebel, Christine; Lévy, Daniel; Jault, Jean-Michel

    2011-01-01

    Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (kcat∼5 s−1). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment. PMID:21602923

  7. Discovering Natural Product Modulators to Overcome Multidrug Resistance in Cancer Chemotherapy

    PubMed Central

    Wu, Chung-Pu; Ohnuma, Shinobu; Ambudkar, Suresh V.

    2012-01-01

    Multidrug resistance caused by the overexpression of ABC drug transporters is a major obstacle in clinical cancer chemotherapy. For several years, it appeared that direct inhibition of ABC transporters would be the cheapest and most efficient way to combat this problem. Unfortunately, progress in finding a potent, selective inhibitor to modulate ABC transporters and restore drug sensitivity in multidrug-resistant cancer cells has been slow and challenging. Candidate drugs should ideally be selective, potent and relatively non-toxic. Many researchers in recent years have turned their attention to utilizing natural products as the building blocks for the development of the next generation of inhibitors, especially after the disappointing results obtained from inhibitors of the first three generations at the clinical trial stage. The first step is to discover natural substances (distinct from the first three generation inhibitors) that are potent, selective and relatively non-toxic in order to be used clinically. Here, we present a brief overview of the prospect of using natural products to modulate the function of ABC drug transporters clinically and their impact on human physiology and pharmacology. PMID:21118092

  8. Lyme Disease-Causing Borrelia Species Encode Multiple Lipoproteins Homologous to Peptide-Binding Proteins of ABC-Type Transporters

    PubMed Central

    Kornacki, Jon A.; Oliver, Donald B.

    1998-01-01

    To identify cell envelope proteins of Borrelia burgdorferi, the causative agent of Lyme disease, we constructed a library of B. burgdorferi genes fused to the Escherichia coli phoA gene, which expresses enzymatically active alkaline phosphatase. One such gene, oppA-1, encodes a predicted polypeptide with significant similarities to various peptide-binding proteins of ABC-type transporters. Immediately downstream of oppA-1 are two genes, oppA-2 and oppA-3, whose predicted polypeptide products show strong similarities in their amino acid sequences to OppA-1, including a sequence that resembles the most highly conserved region in peptide-binding proteins. By labeling with [3H]palmitate, OppA-1, OppA-2, and OppA-3 were shown to be lipoproteins. DNA hybridization analysis showed that the oppA-1 oppA-2 oppA-3 region is located on the linear chromosome of B. burgdorferi, and the genes are conserved among different Borrelia species that cause Lyme disease (B. burgdorferi, B. garinii, and B. afzelli), suggesting that all three homologous genes are important to the maintenance of Lyme disease spirochetes in one or more of their hosts. PMID:9712756

  9. Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1α

    PubMed Central

    Arnason, Terra; Harkness, Troy

    2015-01-01

    Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDR cancers before clinical failure has the potential to offer new approaches to fighting MDR cancer. PMID:26501324

  10. Efflux in the Oral Metagenome: The Discovery of a Novel Tetracycline and Tigecycline ABC Transporter

    PubMed Central

    Reynolds, Liam J.; Roberts, Adam P.; Anjum, Muna F.

    2016-01-01

    Antibiotic resistance in human bacterial pathogens and commensals is threatening our ability to treat infections and conduct common medical procedures. As novel antibiotics are discovered and marketed it is important that we understand how resistance to them may arise and know what environments may act as reservoirs for such resistance genes. In this study a tetracycline and tigecycline resistant clone was identified by screening a human saliva metagenomic library in Escherichia coli EPI300 on agar containing 5 μg/ml tetracycline. Sequencing of the DNA insert present within the tetracycline resistant clone revealed it to contain a 7,765 bp fragment harboring novel ABC half transporter genes, tetAB(60). Mutagenesis studies performed on these genes confirmed that they were responsible for the tetracycline and tigecycline resistance phenotypes. Growth studies performed using E. coli EPI300 clones that harbored either the wild type, the mutated, or none of these genes indicated that there was a fitness cost associated with presence of these genes, with the isolate harboring both genes exhibiting a significantly slower growth than control strains. Given the emergence of E. coli strains that are sensitive only to tigecycline and doxycycline it is concerning that such a resistance mechanism has been identified in the human oral cavity. PMID:27999567

  11. Inactivation of the ABC transporter ATPase gene in Brucella abortus strain 2308 attenuated the virulence of the bacteria.

    PubMed

    Zhang, Min; Han, Xiangan; Liu, Haiwen; Tian, Mingxing; Ding, Chan; Song, Jun; Sun, Xiaoqing; Liu, Zongping; Yu, Shengqing

    2013-06-28

    Brucella abortus is a Gram-negative, facultative intracellular bacterial pathogen of human and other animals. Brucella lipopolysaccharide has been identified as an important virulence factor. In this study, the ABC transporter ATPase gene (BAB1_0542) of B. abortus strain S2308 was inactivated by deleting a 446-bp fragment from the gene, thereby generating the mutant strain, S2308ΔATP. Real time PCR analysis confirmed the inactivation of this gene with no polar effect on the transcription of adjacent genes on the chromosome. The mutant was identified as a rough phenotype strain using heat agglutination test and crystal violet staining. The mutant strain had a different growth rate in Tryptic Soy Broth (TSB), compared to the wild type S2308 strain. Moreover, the mutant strain showed attenuated virulence in vitro and in vivo in RAW264.7 macrophages and Balb/c mice, respectively. Complementation of the mutant strain recovered the smooth phenotype of the bacteria and the complemented strain C2308ΔATP survived for more than four weeks in Balb/c mice, comparable to wild type strain S2308. Furthermore, immunization with the mutant strain protected mice from virulent strain challenge, which suggests the potential for the mutant strain S2308ΔATP as a future vaccine candidate. MHC I, MHC II and co-stimulatory molecule expression levels in mice following infection of S2308ΔATP and S2308 were also investigated.

  12. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management.

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-09-03

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management.

  13. ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells

    PubMed Central

    Boddupalli, Chandra Sekhar; Nair, Shiny; Gray, Simon M.; Nowyhed, Heba N.; Verma, Rakesh; Gibson, Joanna A.; Abraham, Clara; Narayan, Deepak; Vasquez, Juan; Hedrick, Catherine C.; Dhodapkar, Kavita M.; Kaech, Susan M.; Dhodapkar, Madhav V.

    2016-01-01

    Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy. PMID:27617863

  14. Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1.

    PubMed

    Arnason, Terra; Harkness, Troy

    2015-10-16

    Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDRcancers before clinical failure has the potential to offer new approaches to fightingMDRcancer.

  15. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens

    PubMed Central

    Gonzalez, Daniel; Richez, Magali; Bergonzi, Celine; Chabriere, Eric; Elias, Mikael

    2014-01-01

    Phosphate limitation is an important environmental stress that affects the metabolism of various organisms and, in particular, can trigger the virulence of numerous bacterial pathogens. Clostridium perfringens, a human pathogen, is one of the most common causes of enteritis necroticans, gas gangrene and food poisoning. Here, we focused on the high affinity phosphate-binding protein (PBP-1) of an ABC-type transporter, responsible for cellular phosphate uptake. We report the crystal structure (1.65 Å resolution) of the protein in complex with phosphate. Interestingly, PBP-1 does not form the short, low-barrier hydrogen bond with phosphate that is typical of previously characterized phosphate-binding proteins, but rather a canonical hydrogen bond. In its unique binding configuration, PBP-1 forms an unusually high number of hydrogen bonds (14) with the phosphate anion. Discrimination experiments reveal that PBP-1 is the least selective PBP characterised so far and is able to discriminate phosphate from its close competing anion, arsenate, by ~150-fold. PMID:25338617

  16. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management

    PubMed Central

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-01-01

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management. PMID:26333918

  17. An Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants does not endow resistance to Escherichia coli.

    PubMed

    Burris, Kellie; Mentewab, Ayalew; Ripp, Steven; Stewart, C Neal

    2008-03-01

    Concerns have been raised about potential horizontal gene transfer (HGT) of antibiotic resistance markers (ARMs) from transgenic plants to bacteria of medical and environmental importance. All ARMs used in transgenic plants have been bacterial in origin, but it has been recently shown that an Arabidopsis thaliana ABC transporter, Atwbc19, confers kanamycin resistance when overexpressed in transgenic plants. Atwbc19 was evaluated for its ability to transfer kanamycin resistance to Escherichia coli, a kanamycin-sensitive model bacterium, under simulated HGT, staged by subcloning Atwbc19 under the control of a bacterial promoter, genetically transforming to kanamycin-sensitive bacteria, and assessing if resistance was conferred as compared with bacteria harbouring nptII, the standard kanamycin resistance gene used to produce transgenic plants. NptII provided much greater resistance than Atwbc19 and was significantly different from the no-plasmid control at low concentrations. Atwbc19 was not significantly different from the no-plasmid control at higher concentrations. Even though HGT risks are considered low with nptII, Atwbc19 should have even lower risks, as its encoded protein is possibly mistargeted in bacteria.

  18. Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Half molecule ATP-binding cassette transporters of the HMT1(heavy metal tolerance factor 1)subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans and Chlamydomonas reinhardtii, and have homologs in other species, including plants and humans. Based on studies i...

  19. Formation of a Chloride-conducting State in the Maltose ATP-binding Cassette (ABC) Transporter.

    PubMed

    Carlson, Michael L; Bao, Huan; Duong, Franck

    2016-06-03

    ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel.

  20. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

    PubMed Central

    Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

    2016-01-01

    ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

  1. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

    PubMed

    Mann, Evan; Ovchinnikova, Olga G; King, Jerry D; Whitfield, Chris

    2015-10-16

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.

  2. Combination of tenofovir and emtricitabine plus efavirenz: in vitro modulation of ABC transporter and intracellular drug accumulation.

    PubMed

    Bousquet, Laurence; Pruvost, Alain; Guyot, Anne-Cécile; Farinotti, Robert; Mabondzo, Aloïse

    2009-03-01

    Efflux proteins have been shown to greatly affect the uptake of antiretroviral drugs by cells and to hamper their access to the human immunodeficiency virus type 1 replication site. This study evaluated the factors that may lead to drug-drug interactions between emtricitabine (FTC), tenofovir (TFV), and efavirenz (EFV), including the modulation of efflux transporter expression and function. Peripheral blood mononuclear cells from healthy volunteers were used to determine whether or not an interaction between antiretroviral drugs and target cells occurred in any combination of FTC, TFV, EFV, FTC-TFV, TFV-EFV, or FTC-TFV-EFV. Following 20 h of treatment, intracellular drug concentrations were measured by liquid chromatography-tandem mass spectrometry. Efflux transporter functionality and inhibitor drug properties were assessed by measuring fluorescent dye efflux. ABCB1 (P-glycoprotein), ABCC 1 to 6 (multidrug resistance-associated protein), and OAT (organic anion transporter) expression in response to the treatments was quantified by semiquantitative real-time PCR. Cells treated with a double combination (FTC-TFV or TFV-EFV) or the triple combination (FTC-TFV-EFV) produced higher FTC and TFV intracellular concentrations than cells treated with FTC or TFV alone. However, no change in the EFV intracellular concentration was observed. FTC tended to induce abcc5 mRNA expression and EFV tended to induce abcc1 and abcc6 mRNA expression, whereas TFV tended to reduce mdr1, abcc1, abcc5, and abcc6 mRNA expression. Under these conditions, a decrease in the functionality of ABCC was observed, and this decrease was associated with the direct inhibitory actions of these drugs. This in vitro study reveals a benefit of the combination FTC-TFV-EFV in terms of the intracellular FTC and TFV concentrations and highlights the pharmacological mechanisms that lead to this effect.

  3. Expression, purification and structural properties of ABC transporter ABCA4 and its individual domains

    PubMed Central

    Tsybovsky, Yaroslav; Palczewski, Krzysztof

    2014-01-01

    ABCA4 is a member of the A subfamily of ATP-binding cassette transporters that consists of large integral membrane proteins implicated in inherited human diseases. ABCA4 assists in the clearance of N-retinylidene-phosphatidylethanolamine, a potentially toxic by-product of the visual cycle formed in photoreceptors during light perception. Structural and functional studies of this protein have been hindered by its large size, membrane association, and domain complexity. Although mammalian, insect and bacterial systems have been used for expression of ABCA4 and its individual domains, the structural relevance of resulting proteins to the native transporter has yet to be established. We produced soluble domains of ABCA4 in E. coli and S. cerevisiae and the full-length transporter in HEK293 cells. Electron microscopy and size exclusion chromatography were used to assess the conformational homogeneity and structure of these proteins. We found that isolated ABCA4 domains formed large, heterogeneous oligomers cross-linked with non-specific disulphide bonds. Incomplete folding of cytoplasmic domain 2 was proposed based on fluorescence spectroscopy results. In contrast, full-length human ABCA4 produced in mammalian cells was found structurally equivalent to the native protein obtained from bovine photoreceptors. These findings offer recombinantly expressed full-length ABCA4 as an appropriate object for future detailed structural and functional characterization. PMID:24583180

  4. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    PubMed

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides.

  5. Identification of ABC transporter genes in gonad tissue of two Mediterranean sea urchin species: black, Arbacia lixula L., and rocky, Paracentrotus lividus L.

    PubMed

    Bošnjak, Ivana; Zaja, Roko; Klobučar, Roberta Sauerborn; Šver, Lidija; Franekić, Jasna; Smital, Tvrtko

    2013-10-01

    Multixenobiotic resistance (MXR) represents an important cellular detoxification mechanism in aquatic organisms as it provides them robustness toward natural and man-made contaminants. Several ABC transporters have major roles in the MXR phenotype - P-gp/ABCB1, MRP1-3/ABCC1-3 and BCRP/ABCG2. In this study, we identified the presence of ABC transporters involved in the MXR mechanism of Arbacia lixula and Paracentrotus lividus. AlABCB1/P-gp, AlABCC3/MRP3, AlABCC9/SUR-like and AlABCG-like transcripts were identified in A. lixula; and PlABCC1/P-gp, PlABCC3/MRP3, PlABCC5/MRP5, and PlABCC9/SUR-like transcripts in P. lividus. For each of the new partial sequences, we performed detailed phylogenetic and identity analysis as a first step toward full characterization and understanding of the ecotoxicological role of these ABC transporters.

  6. The role of CAPS buffer in expanding the crystallization space of the nucleotide-binding domain of the ABC transporter haemolysin B from Escherichia coli.

    PubMed

    Zaitseva, Jelena; Holland, I Barry; Schmitt, Lutz

    2004-06-01

    Nucleotide-binding domains (NBDs), which are roughly 27 kDa in size, are conserved components of the large family of ABC (ATP-binding cassette) transporters, which includes importers and exporters. NBDs, or ABC-ATPases, supply energy for the translocation of a vast range of substrates across biological membranes. Despite their hydrophilic sequence, many NBDs readily associate in some way with membranes but demonstrate extreme instability in solution upon separation from the complete transporter. Conditions that stabilized the purified ABC domain of the Escherichia coli haemolysin A (HlyA) transporter were developed. This allowed the screening of unlimited crystallization conditions in the presence of different substrates, the performance of reproducible functional assays and the protection of 50 mg ml(-1) protein from precipitation on ice for months. As a result, it became possible to obtain crystals of HlyB-NBD in the presence of ADP and ATP that were suitable for X-ray analysis. Although the focus of these investigations was placed on HlyB-NBD, the strategy described here can be directly transferred to other proteins that display instability in solution.

  7. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, Chih-Chia; Long, Feng; McDermott, Gerry; Shafer, William M.; Yu, Edward W.

    2008-04-01

    The multidrug efflux transporter NorM from N. gonorrhoeae has been crystallized and X-ray diffraction data have been collected to a resolution of 6.5 Å. The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 Å and diffraction data were complete to 6.5 Å resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 Å.

  8. ABC AND SLC TRANSPORTER EXPRESSION AND POT SUBSTRATE CHRACTERIZATION ACROSS THE HUMAN CMEC/D3 BLOOD-BRAIN BARRIER CELL LINE

    PubMed Central

    Carl, Stephen M.; Lindley, David J.; Couraud, Pierre O.; Weksler, Babette B.; Romero, Ignacio; Mowery, Stephanie A.; Knipp, Gregory T.

    2010-01-01

    Purpose 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. Methods 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 Ca2+. Results 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 19Å, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. Conclusion 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

  9. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

    PubMed Central

    2016-01-01

    ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease. PMID:27766264

  10. A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat.

    PubMed

    Krattinger, Simon G; Lagudah, Evans S; Spielmeyer, Wolfgang; Singh, Ravi P; Huerta-Espino, Julio; McFadden, Helen; Bossolini, Eligio; Selter, Liselotte L; Keller, Beat

    2009-03-06

    Agricultural crops benefit from resistance to pathogens that endures over years and generations of both pest and crop. Durable disease resistance, which may be partial or complete, can be controlled by several genes. Some of the most devastating fungal pathogens in wheat are leaf rust, stripe rust, and powdery mildew. The wheat gene Lr34 has supported resistance to these pathogens for more than 50 years. Lr34 is now shared by wheat cultivars around the world. Here, we show that the LR34 protein resembles adenosine triphosphate-binding cassette transporters of the pleiotropic drug resistance subfamily. Alleles of Lr34 conferring resistance or susceptibility differ by three genetic polymorphisms. The Lr34 gene, which functions in the adult plant, stimulates senescence-like processes in the flag leaf tips and edges.

  11. Transport in technicolor: Mapping ATP-binding cassette transporters in sea urchin embryos

    PubMed Central

    Gökirmak, Tufan; Shipp, Lauren E.; Campanale, Joseph P.; Nicklisch, Sascha C.T.; Hamdoun, Amro

    2014-01-01

    One quarter of eukaryotic genes encode membrane proteins. These include nearly 1000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multi-drug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease. PMID:25156004

  12. β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

    PubMed Central

    Coisne, Caroline; Hallier-Vanuxeem, Dorothée; Boucau, Marie-Christine; Hachani, Johan; Tilloy, Sébastien; Bricout, Hervé; Monflier, Eric; Wils, Daniel; Serpelloni, Michel; Parissaux, Xavier; Fenart, Laurence; Gosselet, Fabien

    2016-01-01

    -CDs can significantly reduce the cellular cholesterol content of cells forming atherosclerotic lesions and can subsequently modulate the expression of ABC transporters involved in RCT. The use of methylated β-CDs would represent a valuable and efficient tool to interfere with atherosclerosis pathogenesis in patients, nonetheless their mode of action still needs further investigations to be fully understood and finely controlled at the cellular level. PMID:27252658

  13. The Differential Binding of Antipsychotic Drugs to the ABC Transporter P-Glycoprotein Predicts Cannabinoid-Antipsychotic Drug Interactions.

    PubMed

    Brzozowska, Natalia I; de Tonnerre, Erik J; Li, Kong M; Wang, Xiao Suo; Boucher, Aurelie A; Callaghan, Paul D; Kuligowski, Michael; Wong, Alex; Arnold, Jonathon C

    2017-03-29

    Cannabis use increases rates of psychotic relapse and treatment failure in schizophrenia patients. Clinical studies suggest that cannabis use reduces the efficacy of antipsychotic drugs, but there has been no direct demonstration of this in a controlled study. The present study demonstrates that exposure to the principal phytocannabinoid, Δ(9)-tetrahydrocannabinol (THC), reverses the neurobehavioral effects of the antipsychotic drug risperidone in mice. THC exposure did not influence D2 and 5-HT2A receptor binding, the major targets of antipsychotic action, but it lowered the brain concentrations of risperidone and its active metabolite, 9-hydroxy risperidone. As risperidone and its active metabolite are excellent substrates of the ABC transporter P-glycoprotein (P-gp), we hypothesized that THC might increase P-gp expression at the blood-brain barrier (BBB) and thus enhance efflux of risperidone and its metabolite from brain tissue. We confirmed that the brain disposition of risperidone and 9-hydroxy risperidone is strongly influenced by P-gp, as P-gp knockout mice displayed greater brain concentrations of these drugs than wild-type mice. Furthermore, we demonstrated that THC exposure increased P-gp expression in various brain regions important to risperidone's antipsychotic action. We then showed that THC exposure did not influence the neurobehavioral effects of clozapine. Clozapine shares a very similar antipsychotic mode of action to risperidone, but unlike risperidone is not a P-gp substrate. Our results imply that clozapine or non-P-gp substrate antipsychotic drugs may be better first-line treatments for schizophrenia patients with a history of cannabis use.Neuropsychopharmacology advance online publication, 29 March 2017; doi:10.1038/npp.2017.50.

  14. Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

    PubMed Central

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

    2014-01-01

    Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA 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 Opp system mediates both uptake of peptides and fitness in the respiratory tract. PMID:25156736

  15. Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.

    PubMed

    Dalzell, Abigail M; Mistry, Pratibha; Wright, Jayne; Williams, Faith M; Brown, Colin D A

    2015-06-15

    ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02μM (ivermectin); 0.60±0.07 and 0.56±0.02μM (emamectin) and 0.95±0.08 and 0.77±0.25μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72μM (ivermectin); 1.87±0.57 and 2.74±1.01μM (emamectin) and 2.25±0.01 and 1.68±0.63μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1μM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of

  16. EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae.

    PubMed

    He, Gui-Xin; Thorpe, Conner; Walsh, Dennis; Crow, Robert; Chen, Huizhong; Kumar, Sanath; Varela, Manuel F

    2011-10-01

    We cloned a gene, ECL_03329, from the chromosome of Enterobacter cloacae ATCC13047, using a drug-hypersensitive Escherichia coli KAM32 cell as the host. We show here that this gene, designated as emmdR, is responsible for multidrug resistance in E. cloacae. E. coli KAM32 host cells containing the cloned emmdR gene (KAM32/pEMMDR28) showed decreased susceptibilities to benzalkonium chloride, norfloxacin, ciprofloxacin, levofloxacin, ethidium bromide, acriflavine, rhodamine6G, and trimethoprim. emmdR-deficient E. cloacae cells (EcΔemmdR) showed increased susceptibilities to several of the antimicrobial agents tested. EmmdR has twelve predicted transmembrane segments and some shared identity with members of the multidrug and toxic compound extrusion (MATE) family of transporters. Study of the antimicrobial agent efflux activities revealed that EmmdR is an H+-drug antiporter but not a Na+ driven efflux pump. These results indicate that EmmdR is responsible for multidrug resistance and pumps out quinolones from E. cloacae.

  17. Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant Candida glabrata.

    PubMed

    Gohar, Atefeh Abdollahi; Badali, Hamid; Shokohi, Tahereh; Nabili, Mojtaba; Amirrajab, Nasrin; Moazeni, Maryam

    2017-04-01

    Clinical management of fungal diseases is compromised by the emergence of antifungal drug resistance in fungi, which leads to elimination of available drug classes as treatment options. An understanding of antifungal resistance at molecular level is, therefore, essential for the development of strategies to combat the resistance. This study presents the assessment of molecular mechanisms associated with fluconazole resistance in clinical Candida glabrata isolates originated from Iran. Taking seven distinct fluconazole-resistant C. glabrata isolates, real-time PCRs were performed to evaluate the alternations in the regulation of the genes involved in drug efflux including CgCDR1, CgCDR2, CgSNQ2, and CgERG11. Gain-of-function (GOF) mutations in CgPDR1 alleles were determined by DNA sequencing. Cross-resistance to fluconazole, itraconazole, and voriconazole was observed in 2.5 % of the isolates. In the present study, six amino acid substitutions were identified in CgPdr1, among which W297R, T588A, and F575L were previously reported, whereas D243N, H576Y, and P915R are novel. CgCDR1 overexpression was observed in 57.1 % of resistant isolates. However, CgCDR2 was not co-expressed with CgCDR1. CgSNQ2 was upregulated in 71.4 % of the cases. CgERG11 overexpression does not seem to be associated with azole resistance, except for isolates that exhibited azole cross-resistance. The pattern of efflux pump gene upregulation was associated with GOF mutations observed in CgPDR1. These results showed that drug efflux mediated by adenosine-5-triphosphate (ATP)-binding cassette transporters, especially CgSNQ2 and CgCDR1, is the predominant mechanism of fluconazole resistance in Iranian isolates of C. glabrata. Since some novel GOF mutations were found here, this study also calls for research aimed at investigating other new GOF mutations to reveal the comprehensive understanding about efflux-mediated resistance to azole antifungal agents.

  18. Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

    PubMed Central

    Horlacher, Reinhold; Xavier, Karina B.; Santos, Helena; DiRuggiero, Jocelyne; Kossmann, Marina; Boos, Winfried

    1998-01-01

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a Kd of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches. PMID:9457875

  19. Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) Enhances Multidrug Resistance of Hepatocellular Carcinoma Cells

    PubMed Central

    Fu, Rong-Jie; Lv, Ya-Ping; Jin, Wei; Meng, Chao; Chen, Guo-Qiang; Huang, Lei

    2016-01-01

    China accounts for almost half of the total number of liver cancer cases and deaths worldwide, and hepatocellular carcinoma (HCC) is the most primary liver cancer. Snail family transcriptional repressor 2 (SNAI2) is known as an epithelial to mesenchymal transition-inducing transcription factor that drives neoplastic epithelial cells into mesenchymal phenotype. However, the roles of endogenous SNAI2 remain controversial in different types of malignant tumors. Herein, we surprisingly identify that anchorage-independent growth, including the formation of tumor sphere and soft agar colony, is significantly increased when SNAI2 expression is inhibited by shRNAs in HCC cells. Suppression of SNAI2 suffices to up-regulate several cancer stem genes. Although unrelated to the metastatic ability, SNAI2 inhibition does increase the efflux of Hoechst 33342 and enhance multidrug resistance in vitro and in vivo. In agreement with this data, we demonstrate for the first time that decreasing SNAI2 level can transcriptionally upregulate several ATP binding cassette (ABC) transporter genes such as ABCB1. Moreover, ABC transporters’ inhibitor verapamil can rescue the multidrug resistance induced by SNAI2 inhibition. Our results implicate that SNAI2 behaves as a tumor suppressor by inhibiting multidrug resistance via suppressing ABC transporter genes in HCC cells. PMID:27760172

  20. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells

    PubMed Central

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells. PMID:27536106

  1. Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

    PubMed Central

    Nair, Asha V.; Singh, Himansha; Raturi, Sagar; Neuberger, Arthur; Tong, Zhen; Ding, Ning; Agboh, Kelvin; van Veen, Hendrik W.

    2016-01-01

    The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport. PMID:27917857

  2. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

    PubMed

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

  3. Research Advances: Less Expensive and More Convenient Gaucher's Disease Treatment; Structural Loop Regions: Key to Multidrug-Resistance Transporters?; New Method Identifies Proteins in Old Artwork

    ERIC Educational Resources Information Center

    King, Angela G.

    2006-01-01

    The X-ray structure of EmrD, a multidrug transporter protein from Escherichia coli, common bacteria known to cause several food-borne illnesses was determined by scientists at The Scripps Research Institute. The hydrophobic residues in the EmrD internal cavity are likely to contribute to the general mechanism transporting various compounds through…

  4. Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera.

    PubMed

    Çakır, Birsen; Kılıçkaya, Ozan

    2013-01-01

    The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 "full-size," 41 "half-size," and 15 "soluble" putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.

  5. Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein.

    PubMed

    Hinsa, Shannon M; Espinosa-Urgel, Manuel; Ramos, Juan L; O'Toole, George A

    2003-08-01

    We report the identification of an ATP-binding cassette (ABC) transporter and an associated large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted molecular weight of approximately 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.

  6. Maltose uptake by the novel ABC transport system MusEFGK2I causes increased expression of ptsG in Corynebacterium glutamicum.

    PubMed

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W; Krämer, Reinhard; Seibold, Gerd M

    2013-06-01

    The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [(14)C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum.

  7. Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

    PubMed Central

    Lalloo, Anita K; Luo, Feng R; Guo, Ailan; Paranjpe, Pankaj V; Lee, Sung-Hack; Vyas, Viral; Rubin, Eric; Sinko, Patrick J

    2004-01-01

    Background The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. Methods The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined. Results The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein

  8. Pilocarpine-induced convulsive activity is limited by multidrug transporters at the rodent blood-brain barrier.

    PubMed

    Römermann, K; Bankstahl, J P; Löscher, W; Bankstahl, M

    2015-05-01

    As a result of the growing availability of genetically engineered mouse lines, the pilocarpine post-status epilepticus (SE) model of temporal lobe epilepsy is increasingly used in mice. A discrepancy in pilocarpine sensitivity in FVB/N wild-type versus P-glycoprotein (PGP)-deficient mice precipitated the investigation of the interaction between pilocarpine and two major multidrug transporters at the blood-brain barrier. Doses of pilocarpine necessary for SE induction were determined in male and female wild-type and PGP-deficient mice. Brain and plasma concentrations were measured following low (30-50 mg⋅kg(-1) i.p.) and/or high (200 mg⋅kg(-1) i.p.) doses of pilocarpine in wild-type mice, and mice lacking PGP, breast cancer resistance protein (BCRP), or both transporters, as well as in rats with or without pretreatment with lithium chloride or tariquidar. Concentration equilibrium transport assays (CETA) were performed using cells overexpressing murine PGP or BCRP. Lower pilocarpine doses were necessary for SE induction in PGP-deficient mice. Brain-plasma ratios were higher in mice lacking PGP or PGP and BCRP, which was also observed after pretreatment with tariquidar in mice and in rats. Lithium chloride did not change brain penetration of pilocarpine. CETA confirmed transport of pilocarpine by PGP and BCRP. Pilocarpine is a substrate of PGP and BCRP at the rodent blood-brain barrier, which restricts its convulsive action. Future studies to reveal whether strain differences in pilocarpine sensitivity derive from differences in multidrug transporter expression levels are warranted.

  9. Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees.

    PubMed

    Guseman, Alex J; Miller, Kaliah; Kunkle, Grace; Dively, Galen P; Pettis, Jeffrey S; Evans, Jay D; vanEngelsdorp, Dennis; Hawthorne, David J

    2016-01-01

    Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species.

  10. Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees

    PubMed Central

    Guseman, Alex J.; Miller, Kaliah; Kunkle, Grace; Dively, Galen P.; Pettis, Jeffrey S.; Evans, Jay D.; vanEngelsdorp, Dennis; Hawthorne, David J.

    2016-01-01

    Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species. PMID:26840460

  11. eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

    PubMed Central

    Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

    2017-01-01

    The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

  12. Identification of novel natural inhibitor for NorM - a multidrug and toxic compound extrusion transporter - an insilico molecular modeling and simulation studies.

    PubMed

    Kesherwani, Manish; Michael Gromiha, M; Fukui, Kazuhiko; Velmurugan, Devadasan

    2017-01-01

    The emergence of bacterial multidrug resistance is an increasing problem in treatment of infectious diseases. An important cause for the multidrug resistance of bacteria is the expression of multidrug efflux transporters. The multidrug and toxic compound extrusion (MATE) transporters are most recently recognized as unique efflux system for extrusion of antimicrobials and therapeutic drugs due to energy stored in either Na(+) or H(+) electrochemical gradient. In the present study, high throughput virtual screening of natural compound collections against NorM - a MATE transporter from Neisseria gonorrhea (NorM-NG) has been carried out followed by flexible docking. The molecular simulation in membrane environment has been performed for understanding the stability and binding energetic of top lead compounds. Results identified a compound from the Indian medicinal plant "Terminalia chebula" which has good binding free energy compared to substrates (rhodamine 6 g, ethidium) and more favorable interactions with the central cavity forming active site residues. The compound has restricted movement in TM7, TM8, and TM1, thus blocking the disruption of Na+ - coordination along with equilibrium state bias towards occlude state of NorM transporter. Thus, this compound blocks the effluxing pathway of antimicrobial drugs and provides as a natural bioactive lead inhibitor against NorM transporter in drug-resistant gonorrhea.

  13. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, C.C.; Long, F.; McDermott, G.; Shafer, W.M.; Yu, E.W.

    2008-06-03

    The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 {angstrom} and diffraction data were complete to 6.5 {angstrom} resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 {angstrom}.

  14. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-09

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

  15. ATP-binding cassette transporters in tumor endothelial cells and resistance to metronomic chemotherapy.

    PubMed

    Hida, Kyoko; Kikuchi, Hiroshi; Maishi, Nako; Hida, Yasuhiro

    2017-02-16

    Drug resistance is a major problem in anticancer therapy. ATP-binding cassette (ABC) transporters have a role in the multidrug resistance. A new regimen of chemotherapy has been proposed, called "metronomic chemotherapy". Metronomic chemotherapy is the frequent, regular administration of drug doses designed to maintain low, but active, concentrations of chemotherapeutic drugs over prolonged periods of time, without causing serious toxicities. Metronomic chemotherapy regimens were developed to optimize the antitumor efficacy of agents that target the tumor vasculature instead of tumor cells, and to reduce toxicity of antineoplastic drugs [1]. Nevertheless, recent studies revealed that ABC transporters are expressed at a higher level in the endothelium in the tumor. To avoid resistance to metronomic anti-angiogenic chemotherapy, ABC transporter inhibition of tumor endothelial cells may be a promising strategy. In this mini-review, we discuss the possible mechanism of resistance to metronomic chemotherapy from the viewpoint of tumor endothelial cell biology, focusing on ABC transporters.

  16. Modulation of multidrug resistance protein 1 (MRP1/ABCC1) transport and atpase activities by interaction with dietary flavonoids.

    PubMed

    Leslie, E M; Mao, Q; Oleschuk, C J; Deeley, R G; Cole, S P

    2001-05-01

    The 190-kDa phosphoglycoprotein multidrug resistance protein 1 (MRP1) (ABCC1) confers resistance to a broad spectrum of anticancer drugs and also actively transports certain xenobiotics with reduced glutathione (GSH) (cotransport) as well as conjugated organic anions such as leukotriene C(4) (LTC(4)). In the present study, we have investigated a series of bioflavonoids for their ability to influence different aspects of MRP1 function. Most flavonoids inhibited MRP1-mediated LTC(4) transport in membrane vesicles and inhibition by several flavonoids was enhanced by GSH. Five of the flavonoids were competitive inhibitors of LTC(4) transport (K(i), 2.4-21 microM) in the following rank order of potency: kaempferol > apigenin (+ GSH) > quercetin > myricetin > naringenin (+ GSH). These flavonoids were less effective inhibitors of 17beta-estradiol 17beta-(D-glucuronide) transport. Moreover, their rank order of inhibitory potency for this substrate differed from that for LTC(4) transport inhibition but correlated with their relative lipophilicity. Several flavonoids, especially naringenin and apigenin, markedly stimulated GSH transport by MRP1, suggesting they may be cotransported with this tripeptide. Quercetin inhibited the ATPase activity of purified reconstituted MRP1 but stimulated vanadate-induced trapping of 8-azido-alpha-[(32)P]ADP by MRP1. In contrast, kaempferol and naringenin stimulated both MRP1 ATPase activity and trapping of ADP. In intact MRP1-overexpressing cells, quercetin reduced vincristine resistance from 8.9- to 2.2-fold, whereas kaempferol and naringenin had no effect. We conclude that dietary flavonoids may modulate the organic anion and GSH transport, ATPase, and/or drug resistance-conferring properties of MRP1. However, the activity profile of the flavonoids tested differed from one another, suggesting that at least some of these compounds may interact with different sites on the MRP1 molecule.

  17. Macrophage apolipoprotein A-I expression protects against atherosclerosis in ApoE-deficient mice and up-regulates ABC transporters.

    PubMed

    Su, Yan Ru; Ishiguro, Hiroyuki; Major, Amy S; Dove, Dwayne E; Zhang, Wenwu; Hasty, Alyssa H; Babaev, Vladimir R; Linton, MacRae F; Fazio, Sergio

    2003-10-01

    The antiatherogenic effect of high-density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) has been largely attributed to their key roles in reverse cholesterol transport (RCT) and cellular cholesterol efflux. Substantial evidence shows that overexpression of human apoA-I reduces atherosclerosis in animal models. However, it is uncertain whether this protection is due to an increase in plasma HDL level or to a local effect in the artery wall. To test the hypothesis that expression of human apoA-I in macrophages can promote RCT in the artery wall, we used a retroviral construct expressing human apoA-I cDNA (MFG-HAI) to transduce ApoE(-/-) bone marrow cells and then transplanted these cells into ApoE(-/-) mice with preexisting atherosclerosis. ApoE(-/-) mice reconstituted with MFG-HAI marrow had a significant reduction (30%) in atherosclerotic lesions in the proximal aorta compared to control mice that received marrow expressing MFG parental virus. Peritoneal macrophages isolated from MFG-HAI mice showed a four- to fivefold increase in mRNA expression levels of both ATP-binding cassette (ABC) A1 and ABCG1 compared to controls. Our data demonstrate that gene transfer-mediated expression of human apoA-I in macrophages can compensate in part for apoE deficiency and delay the progression of atherosclerotic lesions by stimulating ABC-dependent cholesterol efflux and RCT.

  18. CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host–parasite boundary in intracellular stages

    PubMed Central

    Perkins, Margaret E.; Riojas, Ynolde A.; Wu, Teresa W.; Le Blancq, Sylvie M.

    1999-01-01

    The intracellular parasite Cryptosporidium parvum develops inside a vacuole at the apex of its epithelial host cell. The developing parasite is separated from the host cell cytoplasm by a zone of attachment that consists of an extensively folded membranous structure known as the feeder organelle. It has been proposed that the feeder organelle is the site of regulation of transport of nutrients and drugs into the parasite. In this report, we localize an ≈200-kDa integral membrane protein, CpABC, from Cryptosporidium parvum to the host–parasite boundary, possibly the feeder organelle. The predicted amino acid sequence of CpABC has significant structural similarity with the cystic fibrosis conductance regulator and the multidrug resistance protein subfamily of ATP-binding cassette proteins. This is an example of a parasite-encoded transport protein localized at the parasite–host interface of an intracellular protozoan. PMID:10318953

  19. Role of organic cation transporter OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2-K for transport and drug interactions of the antiviral lamivudine.

    PubMed

    Müller, Fabian; König, Jörg; Hoier, Eva; Mandery, Kathrin; Fromm, Martin F

    2013-09-15

    The antiviral lamivudine is cleared predominantly by the kidney with a relevant contribution of renal tubular secretion. It is not clear which drug transporters mediate lamivudine renal secretion. Our aim was to investigate lamivudine as substrate of the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins MATE1 and MATE2-K. Uptake experiments were performed in OCT2, MATE1, or MATE2-K single-transfected human embryonic kidney 293 (HEK) cells. Transcellular transport experiments were performed in OCT2 and/or MATE1 single- or double-transfected Madin-Darby canine kidney II (MDCK) cells grown on transwell filters. Lamivudine uptake was significantly increased in HEK-OCT2, HEK-MATE1, and HEK-MATE2-K cells compared to control cells. In transcellular experiments, OCT2 located in the basolateral membrane had no effect on transcellular lamivudine transport. MATE1 located in the apical membrane decreased intracellular concentrations and increased transcellular transport of lamivudine from the basal to the apical compartment. MATE1- or MATE2-K-mediated transport was increased by an oppositely directed pH gradient. Several simultaneously administered drugs inhibited OCT2- or MATE2-K-mediated lamivudine uptake. The strongest inhibitors were carvedilol for OCT2 and trimethoprim for MATE2-K (inhibition by 96.3 and 83.7% at 15 μM, respectively, p<0.001). Trimethoprim inhibited OCT2- and MATE2-K-mediated lamivudine uptake with IC₅₀ values of 13.2 and 0.66 μM, respectively. Transcellular lamivudine transport in OCT2-MATE1 double-transfected cells was inhibited by trimethoprim with an IC₅₀ value of 6.9 μM. Lamivudine is a substrate of renal drug transporters OCT2, MATE1, and MATE2-K. Concomitant administration of drugs that inhibit these transporters could decrease renal clearance of lamivudine.

  20. Prediction of multi-drug resistance transporters using a novel sequence analysis method [version 2; referees: 2 approved

    DOE PAGES

    McDermott, Jason E.; Bruillard, Paul; Overall, Christopher C.; ...

    2015-03-09

    There are many examples of groups of proteins that have similar function, but the determinants of functional specificity may be hidden by lack of sequencesimilarity, or by large groups of similar sequences with different functions. Transporters are one such protein group in that the general function, transport, can be easily inferred from the sequence, but the substrate specificity can be impossible to predict from sequence with current methods. In this paper we describe a linguistic-based approach to identify functional patterns from groups of unaligned protein sequences and its application to predict multi-drug resistance transporters (MDRs) from bacteria. We first showmore » that our method can recreate known patterns from PROSITE for several motifs from unaligned sequences. We then show that the method, MDRpred, can predict MDRs with greater accuracy and positive predictive value than a collection of currently available family-based models from the Pfam database. Finally, we apply MDRpred to a large collection of protein sequences from an environmental microbiome study to make novel predictions about drug resistance in a potential environmental reservoir.« less

  1. Prediction of multi-drug resistance transporters using a novel sequence analysis method [version 2; referees: 2 approved

    SciTech Connect

    McDermott, Jason E.; Bruillard, Paul; Overall, Christopher C.; Gosink, Luke; Lindemann, Stephen R.

    2015-03-09

    There are many examples of groups of proteins that have similar function, but the determinants of functional specificity may be hidden by lack of sequencesimilarity, or by large groups of similar sequences with different functions. Transporters are one such protein group in that the general function, transport, can be easily inferred from the sequence, but the substrate specificity can be impossible to predict from sequence with current methods. In this paper we describe a linguistic-based approach to identify functional patterns from groups of unaligned protein sequences and its application to predict multi-drug resistance transporters (MDRs) from bacteria. We first show that our method can recreate known patterns from PROSITE for several motifs from unaligned sequences. We then show that the method, MDRpred, can predict MDRs with greater accuracy and positive predictive value than a collection of currently available family-based models from the Pfam database. Finally, we apply MDRpred to a large collection of protein sequences from an environmental microbiome study to make novel predictions about drug resistance in a potential environmental reservoir.

  2. Combinatorial Pharmacophore Modeling of Multidrug and Toxin Extrusion Transporter 1 Inhibitors: a Theoretical Perspective for Understanding Multiple Inhibitory Mechanisms

    PubMed Central

    Xu, Yuan; Liu, Xian; Wang, Yulan; Zhou, Nannan; Peng, Jianlong; Gong, Likun; Ren, Jing; Luo, Cheng; Luo, Xiaomin; Jiang, Hualiang; Chen, Kaixian; Zheng, Mingyue

    2015-01-01

    A combinatorial pharmacophore (CP) model for Multidrug and toxin extrusion 1 (MATE1/SLC47A1) inhibitors was developed based on a data set including 881 compounds. The CP model comprises four individual pharmacophore hypotheses, HHR1, DRR, HHR2 and AAAP, which can successfully identify the MATE1 inhibitors with an overall accuracy around 75%. The model emphasizes the importance of aromatic ring and hydrophobicity as two important structural determinants for MATE1 inhibition. Compared with the pharmacophore model of Organic Cation Transporter 2 (OCT2/ SLC22A2), a functional related transporter of MATE1, the hypotheses of AAAP and PRR5 are suggested to be responsible for their ligand selectivity, while HHR a common recognition pattern for their dual inhibition. A series of analysis including molecular sizes of inhibitors matching different hypotheses, matching of representative MATE1 inhibitors and molecular docking indicated that the small inhibitors matching HHR1 and DRR involve in competitive inhibition, while the relatively large inhibitors matching AAAP are responsible for the noncompetitive inhibition by locking the conformation changing of MATE1. In light of the results, a hypothetical model for inhibiting transporting mediated by MATE1 was proposed. PMID:26330298

  3. ATP-binding cassette transporters in reproduction: a new frontier

    PubMed Central

    Bloise, E.; Ortiga-Carvalho, T.M.; Reis, F.M.; Lye, S.J.; Gibb, W.; Matthews, S.G.

    2016-01-01

    BACKGROUND The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as ‘gatekeepers’ at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and

  4. ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4).

    PubMed

    Rius, Maria; Hummel-Eisenbeiss, Johanna; Keppler, Dietrich

    2008-01-01

    The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.

  5. Uncoupled active transport mechanisms accounting for low selectivity in multidrug carriers: P-glycoprotein and SMR antiporters.

    PubMed

    Krupka, R M

    1999-11-15

    The extraordinarily low substrate specificity of P-glycoprotein conflicts with the notion that specific substrate interactions are required in the control of the reaction path in an active transport system. The difficulty is shown to be overcome by a half-coupled mechanism in which the ATP reaction is linked to carrier transformations, as in a fully coupled system, but in which the transported substrate plays a passive role. The mechanism, which requires no specific interaction with the substrate, brings about uphill transport. A half-coupled mechanism is directly supported by two observations: (i) almost completely uncoupled ATPase activity in purified P-glycoprotein, and (ii) a pattern of substrate specificity like that of passive systems, where maximum rates for different substrates vary little (unlike active systems, where maximum rates vary greatly). The mechanism accommodates other findings: partial inhibition of ATPase activity by an actively transported substrate; simultaneous binding and translocation of more than one substrate molecule; and stimulation or inhibition of the transport of one substrate molecule by another. A half-coupled system associated with an internal competitive inhibitor should behave as if tightly coupled, in agreement with the effects of the synthetic peptide, polytryptophan. The degree of coupling in the intact system is yet to be determined, however. A half-coupled ATPase mechanism could originally have evolved in a flippase, where immersion of the carrier in its substrate, the membrane lipid, precludes uncoupled ATP hydrolysis. These concepts may have wider application. An uncoupled antiport mechanism, driven by a proton gradient rather than ATP, can explain low selectivity in the SMR multidrug carriers of bacteria, and a half-coupled mechanism for the ion-driven cotransport of water (the substrate in which the carrier site is immersed) can explain a recently proposed uphill flow of water.

  6. Induction of CYP1A and ABC transporters in European sea bass (Dicentrarchus labrax) upon 2,3,7,8-TCDD waterborne exposure.

    PubMed

    Della Torre, Camilla; Mariottini, Michela; Vannuccini, Maria Luisa; Trisciani, Anna; Marchi, Davide; Corsi, Ilaria

    2014-08-01

    The aim of this study was to characterize the responsiveness of CYP1A and ABC transport proteins in European Sea bass (Dicentrarchus labrax) waterborne exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (46 pg/L) for 24 h and 7 days. Genes modulation (abcb1, abcc1-2, cyp1a), EROD activity were investigated in liver and 2,3,7,8-TCDD bioconcentration in liver and muscle. TCDD induced significantly cyp1a gene expression and EROD activity at 24 h and 7 d. A significant up-regulation of abcb1 was also observed but only after 7 days. No modulation of abcc1 and abcc2 genes was observed. Waterborne TCDD exposure was able to induce CYP1A and abcb1 encoding for P-glycoprotein in juvenile of European sea bass.

  7. Drug uptake pathways of multidrug transporter AcrB studied by molecular simulations and site-directed mutagenesis experiments.

    PubMed

    Yao, Xin-Qiu; Kimura, Nobuhiro; Murakami, Satoshi; Takada, Shoji

    2013-05-22

    Multidrug resistance has been a critical issue in current chemotherapy. In Escherichia coli , a major efflux pump responsible for the multidrug resistance contains a transporter AcrB. Crystallographic studies and mutational assays of AcrB provided much of structural and overall functional insights, which led to the functionally rotating mechanism. However, the drug uptake pathways are somewhat controversial because at least two possible pathways, the vestibule and the cleft paths, were suggested. Here, combining molecular simulations and site-directed mutagenesis experiments, we addressed the uptake mechanism finding that the drug uptake pathways can be significantly different depending on the properties of drugs. First, in the computational free energy analysis of drug movements along AcrB tunnels, we found a ligand-dependent drug uptake mechanism. With the same molecular sizes, drugs that are both strongly hydrophobic and lipophilic were preferentially taken in via the vestibule path, while other drugs favored the cleft path. Second, direct simulations realized totally about 3500 events of drug uptake by AcrB for a broad range of drug property. These simulations confirmed the ligand-dependent drug uptake and further suggested that a smaller drug favors the vestibule path, while a larger one is taken in via the cleft path. Moreover, the direct simulations identified an alternative uptake path which is not visible in the crystal structure. Third, site-directed mutagenesis of AcrB in E. coli verified that mutations of residues located along the newly identified path significantly reduced the efflux efficiency, supporting its relevance in in vivo function.

  8. Human ABCG2: structure, function, and its role in multidrug resistance

    PubMed Central

    Mo, Wei; Zhang, Jian-Ting

    2012-01-01

    Human ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily and is known to contribute to multidrug resistance (MDR) in cancer chemotherapy. Among ABC transporters that are known to cause MDR, ABCG2 is particularly interesting for its potential role in protecting cancer stem cells and its complex oligomeric structure. Recent studies have also revealed that the biogenesis of ABCG2 could be modulated by small molecule compounds. These modulators, upon binding to ABCG2, accelerate the endocytosis and trafficking to lysosome for degradation and effectively reduce the half-life of ABCG2. Hence, targeting ABCG2 stability could be a new venue for therapeutic discovery to sensitize drug resistant human cancers. In this report, we review recent progress on understanding the structure, function, biogenesis, as well as physiological and pathophysiological functions of ABCG2. PMID:22509477

  9. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    PubMed

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  10. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed Central

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-01-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

  11. Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry ▿ †

    PubMed Central

    Boucher, Nathalie; Noll, Kenneth M.

    2011-01-01

    The chromosome of Thermotoga maritima strain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound with Kd (dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars: T. maritima MSB8 genomovar TIGR and T. maritima MSB8 genomovar DSM 3109, respectively. PMID:21764944

  12. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-04-01

    Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella.

  13. 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...

  14. Bacillus cereus efflux protein BC3310 – a multidrug transporter of the unknown major facilitator family, UMF-2

    PubMed Central

    Kroeger, Jasmin K.; Hassan, Karl; Vörös, Aniko; Simm, Roger; Saidijam, Massoud; Bettaney, Kim E.; Bechthold, Andreas; Paulsen, Ian T.; Henderson, Peter J. F.; Kolstø, Anne-Brit

    2015-01-01

    Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria. PMID:26528249

  15. Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity.

    PubMed

    Esser, Lothar; Zhou, Fei; Pluchino, Kristen M; Shiloach, Joseph; Ma, Jichun; Tang, Wai-Kwan; Gutierrez, Camilo; Zhang, Alex; Shukla, Suneet; Madigan, James P; Zhou, Tongqing; Kwong, Peter D; Ambudkar, Suresh V; Gottesman, Michael M; Xia, Di

    2017-01-13

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

  16. Glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth in human cells.

    PubMed

    Hong, Yifan; Lai, Yau-Tsz; Chan, Godfrey Chi-Fung; Sun, Hongzhe

    2015-03-17

    Glutathione and multidrug resistance protein (MRP) play an important role on the metabolism of a variety of drugs. Bismuth drugs have been used to treat gastrointestinal disorder and Helicobacter pylori infection for decades without exerting acute toxicity. They were found to interact with a wide variety of biomolecules, but the major metabolic pathway remains unknown. For the first time (to our knowledge), we systematically and quantitatively studied the metabolism of bismuth in human cells. Our data demonstrated that over 90% of bismuth was passively absorbed, conjugated to glutathione, and transported into vesicles by MRP transporter. Mathematical modeling of the system reveals an interesting phenomenon. Passively absorbed bismuth consumes intracellular glutathione, which therefore activates de novo biosynthesis of glutathione. Reciprocally, sequestration by glutathione facilitates the passive uptake of bismuth and thus completes a self-sustaining positive feedback circle. This mechanism robustly removes bismuth from both intra- and extracellular space, protecting critical systems of human body from acute toxicity. It elucidates the selectivity of bismuth drugs between human and pathogens that lack of glutathione, such as Helicobacter pylori, opening new horizons for further drug development.

  17. Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity*♦

    PubMed Central

    Esser, Lothar; Zhou, Fei; Pluchino, Kristen M.; Shiloach, Joseph; Ma, Jichun; Tang, Wai-kwan; Gutierrez, Camilo; Zhang, Alex; Shukla, Suneet; Madigan, James P.; Zhou, Tongqing; Kwong, Peter D.; Ambudkar, Suresh V.; Gottesman, Michael M.; Xia, Di

    2017-01-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions. PMID:27864369

  18. Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Kai; Zhang, Guan-Nan; Wang, Yi-Jun; Patel, Bhargav A.; Talele, Tanaji T.; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-05-01

    ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [3H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [3H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 μM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment.

  19. Glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth in human cells

    PubMed Central

    Hong, Yifan; Lai, Yau-Tsz; Chan, Godfrey Chi-Fung; Sun, Hongzhe

    2015-01-01

    Glutathione and multidrug resistance protein (MRP) play an important role on the metabolism of a variety of drugs. Bismuth drugs have been used to treat gastrointestinal disorder and Helicobacter pylori infection for decades without exerting acute toxicity. They were found to interact with a wide variety of biomolecules, but the major metabolic pathway remains unknown. For the first time (to our knowledge), we systematically and quantitatively studied the metabolism of bismuth in human cells. Our data demonstrated that over 90% of bismuth was passively absorbed, conjugated to glutathione, and transported into vesicles by MRP transporter. Mathematical modeling of the system reveals an interesting phenomenon. Passively absorbed bismuth consumes intracellular glutathione, which therefore activates de novo biosynthesis of glutathione. Reciprocally, sequestration by glutathione facilitates the passive uptake of bismuth and thus completes a self-sustaining positive feedback circle. This mechanism robustly removes bismuth from both intra- and extracellular space, protecting critical systems of human body from acute toxicity. It elucidates the selectivity of bismuth drugs between human and pathogens that lack of glutathione, such as Helicobacter pylori, opening new horizons for further drug development. PMID:25737551

  20. The Hedgehog receptor patched functions in multidrug transport and chemotherapy resistance.

    PubMed

    Bidet, Michel; Tomico, Amandine; Martin, Patrick; Guizouarn, Hélène; Mollat, Patrick; Mus-Veteau, Isabelle

    2012-11-01

    Most anticancer drugs fail to eradicate tumors, leading to the development of drug resistance and disease recurrence. The Hedgehog signaling plays a crucial role during embryonic development, but is also involved in cancer development, progression, and metastasis. The Hedgehog receptor Patched (Ptc) is a Hedgehog signaling target gene that is overexpressed in many cancer cells. Here, we show a link between Ptc and resistance to chemotherapy, and provide new insight into Ptc function. Ptc is cleared from the plasma membrane upon interaction with its ligand Hedgehog, or upon treatment of cells with the Hedgehog signaling antagonist cyclopamine. In both cases, after incubation of cells with doxorubicin, a chemotherapeutic agent that is used for the clinical management of recurrent cancers, we observed an inhibition of the efflux of doxorubicin from Hedgehog-responding fibroblasts, and an increase of doxorubicin accumulation in two different cancer cell lines that are known to express aberrant levels of Hedgehog signaling components. Using heterologous expression system, we stringently showed that the expression of human Ptc conferred resistance to growth inhibition by several drugs from which chemotherapeutic agents such as doxorubicin, methotrexate, temozolomide, and 5-fluorouracil. Resistance to doxorubicin correlated with Ptc function, as shown using mutations from Gorlin's syndrome patients in which the Ptc-mediated effect on Hedgehog signaling is lost. Our results show that Ptc is involved in drug efflux and multidrug resistance, and suggest that Ptc contributes to chemotherapy resistance of cancer cells.

  1. Coarse-grained Simulations of Conformational Changes in Multidrug Resistance Transporters

    NASA Astrophysics Data System (ADS)

    Jewel, S. M. Yead; Dutta, Prashanta; Liu, Jin

    2016-11-01

    The overexpression of multidrug resistance (MDR) systems on the gram negative bacteria causes serious problems for treatment of bacterial infectious diseases. The system effectively pumps the antibiotic drugs out of the bacterial cells. During the pumping process one of the MDR components, AcrB undergoes a series of large-scale conformational changes which are responsible for drug recognition, binding and expelling. All-atom simulations are unable to capture those conformational changes because of computational cost. Here, we implement a hybrid coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid, to investigate the proton-dependent conformational changes of AcrB. The simulation results in early stage ( 100 ns) of proton-dependent conformational changes agree with all-atom simulations, validating the coarse-grained model. The coarse-grained force field allows us to explore the process in microsecond simulations. Starting from the crystal structures of Access(A)/Binding(B)/Extrusion(E) monomers in AcrB, we find that deprotonation of Asp407 and Asp408 in monomer E causes a series of large-scale conformational changes from ABE to AAA in absence of drug molecules, which is consistent with experimental findings. This work is supported by NIH Grant: 1R01GM122081-01.

  2. Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

    PubMed Central

    Martell, R. L.; Slapak, C. A.; Levy, S. B.

    1997-01-01

    The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

  3. RNA-Seq Analysis of the Effect of Kanamycin and the ABC Transporter AtWBC19 on Arabidopsis thaliana Seedlings Reveals Changes in Metal Content

    PubMed Central

    Mentewab, Ayalew; Matheson, Kinnari; Adebiyi, Morayo; Robinson, Shanice; Elston, Brianna

    2014-01-01

    Plants are exposed to antibiotics produced by soil microorganisms, but little is known about their responses at the transcriptional level. Likewise, few endogenous mechanisms of antibiotic resistance have been reported. The Arabidopsis thaliana ATP Binding Cassette (ABC) transporter AtWBC19 (ABCG19) is known to confer kanamycin resistance, but the exact mechanism of resistance is not well understood. Here we examined the transcriptomes of control seedlings and wbc19 mutant seedlings using RNA-seq analysis. Exposure to kanamycin indicated changes in the organization of the photosynthetic apparatus, metabolic fluxes and metal uptake. Elemental analysis showed a 60% and 80% reduction of iron uptake in control and wbc19 mutant seedlings respectively, upon exposure to kanamycin. The drop in iron content was accompanied by the upregulation of the gene encoding for FERRIC REDUCTION OXIDASE 6 (FRO6) in mutant seedlings but not by the differential expression of other transport genes known to be induced by iron deficiency. In addition, wbc19 mutants displayed a distinct expression profile in the absence of kanamycin. Most notably the expression of several zinc ion binding proteins, including ZINC TRANSPORTER 1 PRECURSOR (ZIP1) was increased, suggesting abnormal zinc uptake. Elemental analysis confirmed a 50% decrease of zinc content in wbc19 mutants. Thus, the antibiotic resistance gene WBC19 appears to also have a role in zinc uptake. PMID:25310285

  4. RNA-seq analysis of the effect of kanamycin and the ABC transporter AtWBC19 on Arabidopsis thaliana seedlings reveals changes in metal content.

    PubMed

    Mentewab, Ayalew; Matheson, Kinnari; Adebiyi, Morayo; Robinson, Shanice; Elston, Brianna

    2014-01-01

    Plants are exposed to antibiotics produced by soil microorganisms, but little is known about their responses at the transcriptional level. Likewise, few endogenous mechanisms of antibiotic resistance have been reported. The Arabidopsis thaliana ATP Binding Cassette (ABC) transporter AtWBC19 (ABCG19) is known to confer kanamycin resistance, but the exact mechanism of resistance is not well understood. Here we examined the transcriptomes of control seedlings and wbc19 mutant seedlings using RNA-seq analysis. Exposure to kanamycin indicated changes in the organization of the photosynthetic apparatus, metabolic fluxes and metal uptake. Elemental analysis showed a 60% and 80% reduction of iron uptake in control and wbc19 mutant seedlings respectively, upon exposure to kanamycin. The drop in iron content was accompanied by the upregulation of the gene encoding for FERRIC REDUCTION OXIDASE 6 (FRO6) in mutant seedlings but not by the differential expression of other transport genes known to be induced by iron deficiency. In addition, wbc19 mutants displayed a distinct expression profile in the absence of kanamycin. Most notably the expression of several zinc ion binding proteins, including ZINC TRANSPORTER 1 PRECURSOR (ZIP1) was increased, suggesting abnormal zinc uptake. Elemental analysis confirmed a 50% decrease of zinc content in wbc19 mutants. Thus, the antibiotic resistance gene WBC19 appears to also have a role in zinc uptake.

  5. Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

    PubMed

    Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

    2015-01-01

    The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In

  6. Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

    PubMed Central

    Martorana, Alessandra M.; Benedet, Mattia; Maccagni, Elisa A.; Sperandeo, Paola; Villa, Riccardo; Dehò, Gianni

    2016-01-01

    ABSTRACT The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCE The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a

  7. A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

    PubMed

    Davison, Jack R; Lohith, Katheryn M; Wang, Xiaoning; Bobyk, Kostyantyn; Mandadapu, Sivakoteswara R; Lee, Su-Lin; Cencic, Regina; Nelson, Justin; Simpkins, Scott; Frank, Karen M; Pelletier, Jerry; Myers, Chad L; Piotrowski, Jeff; Smith, Harold E; Bewley, Carole A

    2017-04-03

    The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity, but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold improved potency against a range of laboratory and clinical bacterial strains, which we named P10. Whole genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that the restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds including fluoroquinolone antibiotics.

  8. The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma

    PubMed Central

    Pessina, Sara; Cantini, Gabriele; Kapetis, Dimos; Cazzato, Emanuela; Di Ianni, Natalia; Finocchiaro, Gaetano; Pellegatta, Serena

    2016-01-01

    ABSTRACT Abcc3, a member of the ATP-binding cassette transporter superfamily, plays a role in multidrug resistance. Here, we found that Abcc3 is highly expressed in blood-derived NK cells but not in CD8+ T cells. In GL261 glioma-bearing mice treated with the alkylating agent temozolomide (TMZ) for 5 d, an early increased frequency of NK cells was observed. We also found that Abcc3 is strongly upregulated and functionally active in NK cells from mice treated with TMZ compared to controls. We demonstrate that Abcc3 is critical for NK cell survival during TMZ administration; more importantly, Akt, involved in lymphocyte survival, is phosphorylated only in NK cells expressing Abcc3. The resistance of NK cells to chemotherapy was accompanied by increased migration and homing in the brain at early time points. Cytotoxicity, evaluated by IFNγ production and specific lytic activity against GL261 cells, increased peripherally in the later phases, after conclusion of TMZ treatment. Intra-tumor increase of the NK effector subset as well as in IFNγ, granzymes and perforin-1 expression, were found early and persisted over time, correlating with a profound modulation on glioma microenvironment induced by TMZ. Our findings reveal an important involvement of Abcc3 in NK cell resistance to chemotherapy and have important clinical implications for patients treated with chemo-immunotherapy. PMID:27467914

  9. 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.

  10. A Transcriptional Regulator and ABC Transporters Link Stress Tolerance, (p)ppGpp, and Genetic Competence in Streptococcus mutans▿ †

    PubMed Central

    Seaton, Kinda; Ahn, Sang-Joon; Sagstetter, Ann M.; Burne, Robert A.

    2011-01-01

    Streptococcus mutans, a primary agent of dental caries, has three (p)ppGpp synthases: RelA, which is required for a mupirocin-induced stringent response; RelP, which produces (p)ppGpp during exponential growth and is regulated by the RelRS two-component system; and RelQ. Transcription of relPRS and a gene cluster (SMu0835 to SMu0837) located immediately upstream was activated in cells grown with aeration and during a stringent response, respectively. Bioinformatic analysis predicted that SMu0836 and SMu0837 encode ABC exporters, which we designated rcrPQ (rel competence-related) genes, respectively. SMu0835 (rcrR) encodes a MarR family transcriptional regulator. Reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR analysis showed that RcrR functions as an autogenous negative regulator of the expression of the rcrRPQ operon. A mutant in which a polar insertion replaced the SMu836 gene (Δ836polar) grew more slowly and had final yields that were lower than those of the wild-type strain. Likewise, the Δ836polar strain had an impaired capacity to form biofilms, grew poorly at pH 5.5, and was more sensitive to oxidative stressors. Optimal expression of rcrPQ required RelP and vice versa. Replacement of rcrR with a nonpolar antibiotic resistance marker (Δ835np), which leads to overexpression of rcrPQ, yielded a strain that was not transformable with exogenous DNA. Transcriptional analysis revealed that the expression of comYA and comX was dramatically altered in the Δ835np and Δ836polar mutants. Collectively, the data support the suggestion that the rcrRPQ gene products play a critical role in physiologic homeostasis and stress tolerance by linking (p)ppGpp metabolism, acid and oxidative stress tolerance, and genetic competence. PMID:21148727

  11. Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    PubMed Central

    Miyakoshi, Masatoshi; Chao, Yanjie; Vogel, Jörg

    2015-01-01

    There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the gltIJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the gltIJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources. PMID:25630703

  12. Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station

    PubMed Central

    Sippel, K. H.; Bacik, J.; Quiocho, F. A.; Fisher, S. Z.

    2014-01-01

    Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4 −) and dibasic (HPO4 2−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3 were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily. PMID:24915101

  13. The Half-Size ABC Transporter FOLDED PETALS 2/ABCG13 Is Involved in Petal Elongation through Narrow Spaces in Arabidopsis thaliana Floral Buds.

    PubMed

    Takeda, Seiji; Iwasaki, Akira; Tatematsu, Kiyoshi; Okada, Kiyotaka

    2014-08-15

    Flowers are vital for attracting pollinators to plants and in horticulture for humans. Petal morphogenesis is a central process of floral development. Petal development can be divided into three main processes: the establishment of organ identity in a concentric pattern, primordia initiation at fixed positions within a whorl, and morphogenesis, which includes petal elongation through the narrow spaces within the bud. Here, we show that the FOLDED PETALS 2 (FOP2) gene, encoding a member of the half-size ATP binding cassette (ABC) transporter family ABCG13, is involved in straight elongation of petals in Arabidopsis thaliana. In fop2 mutants, flowers open with folded petals, instead of straight-elongated ones found in the wild type. The epicuticular nanoridge structures are absent in many abaxial epidermal cells of fop2 petals, and surgical or genetic generation of space in young fop2 buds restores the straight elongation of petals, suggesting that the physical contact of sepals and petals causes the petal folding. Similar petal folding has been reported in the fop1 mutant, and the petals of fop2 fop1 double mutants resemble those of both the fop1 and fop2 single mutants, although the epidermal structure and permeability of the petal surface is more affected in fop2. Our results suggest that synthesis and transport of cutin or wax in growing petals play an important role for their smooth elongation through the narrow spaces of floral buds.

  14. Urinary Dopamine as a Potential Index of the Transport Activity of Multidrug and Toxin Extrusion in the Kidney.

    PubMed

    Kajiwara, Moto; Ban, Tsuyoshi; Matsubara, Kazuo; Nakanishi, Yoichi; Masuda, Satohiro

    2016-07-30

    Dopamine is a cationic natriuretic catecholamine synthesized in proximal tubular cells (PTCs) of the kidney before secretion into the lumen, a key site of its action. However, the molecular mechanisms underlying dopamine secretion into the lumen remain unclear. Multidrug and toxin extrusion (MATE) is a H⁺/organic cation antiporter that is highly expressed in the brush border membrane of PTCs and mediates the efflux of organic cations, including metformin and cisplatin, from the epithelial cells into the urine. Therefore, we hypothesized that MATE mediates dopamine secretion, a cationic catecholamine, into the tubule lumen, thereby regulating natriuresis. Here, we show that [³H]dopamine uptake in human (h) MATE1-, hMATE-2K- and mouse (m) MATE-expressing cells exhibited saturable kinetics. Fluid retention and decreased urinary excretion of dopamine and Na⁺ were observed in Mate1-knockout mice compared to that in wild-type mice. Imatinib, a MATE inhibitor, inhibited [³H]dopamine uptake by hMATE1-, hMATE2-K- and mMATE1-expressing cells in a concentration-dependent manner. At clinically-relevant concentrations, imatinib inhibited [³H]dopamine uptake by hMATE1- and hMATE2-K-expressing cells. The urinary excretion of dopamine and Na⁺ decreased and fluid retention occurred in imatinib-treated mice. In conclusion, MATE transporters secrete renally-synthesized dopamine, and therefore, urinary dopamine has the potential to be an index of the MATE transporter activity.

  15. ABCG15 encodes an ABC transporter protein, and is essential for post-meiotic anther and pollen exine development in rice.

    PubMed

    Qin, Peng; Tu, Bin; Wang, Yuping; Deng, Luchang; Quilichini, Teagen D; Li, Ting; Wang, Hui; Ma, Bingtian; Li, Shigui

    2013-01-01

    In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers.

  16. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

    PubMed

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  17. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

    SciTech Connect

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  18. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine.

    PubMed

    Drozdzik, Marek; Gröer, Christian; Penski, Jette; Lapczuk, Joanna; Ostrowski, Marek; Lai, Yurong; Prasad, Bhagwat; Unadkat, Jashvant D; Siegmund, Werner; Oswald, Stefan

    2014-10-06

    Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

  19. Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

    PubMed

    Elsby, Robert; Smith, Veronica; Fox, Lisa; Stresser, David; Butters, Caroline; Sharma, Pradeep; Surry, Dominic D

    2011-09-01

    Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

  20. Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR.

    PubMed

    Terán, Wilson; Felipe, Antonia; Segura, Ana; Rojas, Antonia; Ramos, Juan-Luis; Gallegos, María-Trinidad

    2003-10-01

    Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics. In P. putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol. We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process. ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline. In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline. These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents. This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.

  1. Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex.

    PubMed

    Kim, Yeon-Joo; Kim, Ji-Eun; Choi, Hui-Chul; Song, Hong-Ki; Kang, Tae-Cheon

    2015-06-01

    In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy.

  2. Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.

    PubMed

    Garvey, Mark I; Baylay, Alison J; Wong, Ryan L; Piddock, Laura J V

    2011-01-01

    Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance.

  3. Differential expression of peroxidase and ABC transporter as the key regulatory components for degradation of azo dyes by Penicillium oxalicum SAR-3.

    PubMed

    Saroj, Samta; Kumar, Karunesh; Prasad, Manoj; Singh, R P

    2014-12-01

    Fungal species are potential dye decomposers since these secrete spectra of extracellular enzymes involved in catabolism. However, cellular mechanisms underlying azo dye catalysis and detoxification are incompletely understood and obscure. A potential strain designated as Penicillium oxalicum SAR-3 demonstrated broad-spectrum catabolic ability of different azo dyes. A forward suppression subtractive hybridization (SSH) cDNA library of P. oxalicum SAR-3 constructed in presence and absence of azo dye Acid Red 183 resulted in identification of 183 unique expressed sequence tags (ESTs) which were functionally classified into 12 functional categories. A number of novel genes that affect specifically organic azo dye degradation were discovered. Although the ABC transporters and peroxidases emerged as prominent hot spot for azo dye detoxification, we also identified a number of proteins that are more proximally related to stress-responsive gene expression. Majority of the ESTs (29.5%) were grouped as hypothetical/unknown indicating the presence of putatively novel genes. Analysis of few ESTs through quantitative real-time reverse transcription polymerase chain reaction revealed their possible role in AR183 degradation. The ESTs identified in the SSH library provide a novel insight on the transcripts that are expressed in P. oxalicum strain SAR-3 in response to AR183.

  4. Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation.

    PubMed

    Currie, Erin; King, Brian; Lawrenson, Andrea L; Schroeder, Lena K; Kershner, Aaron M; Hermann, Greg J

    2007-11-01

    Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(-) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.

  5. Vandetanib (Zactima, ZD6474) Antagonizes ABCC1- and ABCG2-Mediated Multidrug Resistance by Inhibition of Their Transport Function

    PubMed Central

    Zheng, Li-sheng; Wang, Fang; Li, Yu-hong; Zhang, Xu; Chen, Li-ming; Liang, Yong-ju; Dai, Chun-ling; Yan, Yan-yan; Tao, Li-yang; Mi, Yan-jun; Yang, An-kui; To, Kenneth Kin Wah; Fu, Li-wu

    2009-01-01

    Background ABCC1 and ABCG2 are ubiquitous ATP-binding cassette transmembrane proteins that play an important role in multidrug resistance (MDR). In this study, we evaluated the possible interaction of vandetanib, an orally administered drug inhibiting multiple receptor tyrosine kinases, with ABCC1 and ABCG2 in vitro. Methodology and Principal Findings MDR cancer cells overexpressing ABCC1 or ABCG2 and their sensitive parental cell lines were used. MTT assay showed that vandetanib had moderate and almost equal-potent anti-proliferative activity in both sensitive parental and MDR cancer cells. Concomitant treatment of MDR cells with vandetanib and specific inhibitors of ABCC1 or ABCG2 did not alter their sensitivity to the former drug. On the other hand, clinically attainable but non-toxic doses of vandetanib were found to significantly enhance the sensitivity of MDR cancer cells to ABCC1 or ABCG2 substrate antitumor drugs. Flow cytometric analysis showed that vandetanib treatment significantly increase the intracellular accumulation of doxorubicin and rhodamine 123, substrates of ABCC1 and ABCG2 respectively, in a dose-dependent manner (P<0.05). However, no significant effect was shown in sensitive parental cell lines. Reverse transcription-PCR and Western blot analysis showed that vandetanib did not change the expression of ABCC1 and ABCG2 at both mRNA and protein levels. Furthermore, total and phosphorylated forms of AKT and ERK1/2 remained unchanged after vandetanib treatment in both sensitive and MDR cancer cells. Conclusions Vandetanib is unlikely to be a substrate of ABCC1 or ABCG2. It overcomes ABCC1- and ABCG2-mediated drug resistance by inhibiting the transporter activity, independent of the blockade of AKT and ERK1/2 signal transduction pathways. PMID:19390592

  6. Compromised in vitro dissolution and membrane transport of multidrug amorphous formulations.

    PubMed

    Alhalaweh, Amjad; Bergström, Christel A S; Taylor, Lynne S

    2016-05-10

    Herein, the thermodynamic properties of solutions evolving from the non-sink dissolution of amorphous solid dispersions (ASDs) containing two or more drugs have been evaluated, focusing on the maximum achievable supersaturation and tendency of the system to undergo liquid-liquid phase separation (LLPS). Ritonavir (RTV) and atazanavir (ATV) were co-formulated with polyvinylpyrrolidone to produce ASDs with different molar ratios of each drug, and the dissolution profile of each drug was studied under non-sink conditions. The phase behavior of the supersaturated solutions generated by ASD dissolution was compared to that of supersaturated solutions generated by antisolvent addition. Dissolution of an ASD containing RTV, ATV and lopinavir (LPV) was also investigated. A thermodynamic model was used to predict the maximum achievable supersaturation for ASDs containing two and three drugs. In addition, a transport study with Caco-2 cells was conducted to evaluate the impact of co-addition of drugs on membrane transport. It was found that the formulation containing a 1:1 molar ratio of RTV and ATV achieved only 50% of the supersaturation attained by dissolution of the single drug systems. The maximum achievable concentration of ATV decreased linearly as the mole fraction of ATV in the formulation decreased and a similar trend was observed for RTV. For the dispersion containing a 1:1:1 molar ratio of RTV, ATV and LPV, the maximum concentration of each drug was only one third of that achieved for the single drug formulations. The decrease in the achievable supersaturation was well-predicted by the thermodynamic model for both the binary and ternary drug combinations. These observations can be explained by a decrease in the concentration at which the drugs undergo LLPS in the presence of other miscible drugs, thereby reducing the maximum achievable supersaturation of each drug. The reduced free drug concentration was reflected by a decreased flux across Caco-2 cells for the

  7. Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism.

    PubMed

    Jebbar, Mohamed; Sohn-Bösser, Linda; Bremer, Erhard; Bernard, Théophile; Blanco, Carlos

    2005-02-01

    To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

  8. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    PubMed

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  9. Proton-dependent multidrug efflux systems.

    PubMed Central

    Paulsen, I T; Brown, M H; Skurray, R A

    1996-01-01

    Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also

  10. Mutations of the multi-drug resistance-associated protein ABC transporter gene 5 result in reduction of phytic acid in rice seeds.

    PubMed

    Xu, Xiu-Hong; Zhao, Hai-Jun; Liu, Qing-Long; Frank, Thomas; Engel, Karl-Heinz; An, Gynheung; Shu, Qing-Yao

    2009-06-01

    Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is important to the nutritional quality of cereal and legume seeds. PA and its salts with micronutrient cations, such as iron and zinc, cannot be digested by humans and non-ruminant animals, and hence may affect food/feed nutritional value and cause P pollution of groundwater from animal waste. We previously developed a set of low phytic acid (LPA) rice mutant lines with the aim of increasing the nutritional quality of rice. Two of these lines, Os-lpa-XS110-2 (homozygous non-lethal) Os-lpa-XS110-3 (homozygous lethal), contain two mutant alleles of a LPA gene (hereafter XS-lpa2-1 and XS-lpa2-2, respectively). In this study, we mapped the XS-lpa2-1 gene to a region on chromosome 3 between microsatellite markers RM14360 and RM1332, where the rice orthologue (OsMRP5) of the maize lpa1 gene is located. Sequence analysis of the OsMRP5 gene revealed a single base pair change (C/G-T/A transition) in the sixth exon of XS-lpa2-1 and a 5-bp deletion in the first exon of XS-lpa2-2. OsMRP5 is expressed in both vegetative tissues and developing seeds, and the two mutations do not change the level of RNA transcription. A T-DNA insertion line, 4A-02500, in which OsMRP5 was disrupted, also showed the same high inorganic phosphorus phenotype as Os-lpa-XS110-3 and appeared to be homozygous lethal. PA is significantly reduced in Os-lpa-XS110-2 (~20%) and in 4A-02500 (~90%) seeds compared with their wild type lines, and no PA was detected in Os-lpa-XS110-3 using HPLC analysis. This evidence indicates that the OsMRP5 gene plays an important role in PA metabolism in rice seeds.

  11. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    PubMed Central

    Richmond, Grace E.; Evans, Laura P.; Anderson, Michele J.; Wand, Matthew E.; Bonney, Laura C.; Ivens, Alasdair; Chua, Kim Lee; Webber, Mark A.; Sutton, J. Mark; Peterson, Marnie L.

    2016-01-01

    ABSTRACT The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery. PMID:27094331

  12. Water-mediated forces between the nucleotide binding domains generate the power stroke in an ABC transporter

    NASA Astrophysics Data System (ADS)

    Furukawa-Hagiya, Tomoka; Yoshida, Norio; Chiba, Shuntaro; Hayashi, Tomohiko; Furuta, Tadaomi; Sohma, Yoshiro; Sakurai, Minoru

    2014-11-01

    ATP binding cassette proteins shuttle a variety of molecules across cell membranes. The substrate transportation process is initiated by the ATP-driven dimerization of nucleotide binding domains (NBDs). Here, the integral-equation theory of liquids was applied to simulated NBD structures to analyze their dimerization process from the viewpoint of thermodynamics and the water-mediated interaction between the NBDs. It was found that a long-range hydration force of enthalpic origin drives the two NBDs to approach from a large separation. In the subsequent step, the water-mediated attraction of entropic origin brings about a structural adjustment between the two NBDs and their tighter contact.

  13. High-throughput screening of dipeptide utilization mediated by the ABC transporter DppBCDF and its substrate-binding proteins DppA1-A5 in Pseudomonas aeruginosa.

    PubMed

    Pletzer, Daniel; Lafon, Corinne; Braun, Yvonne; Köhler, Thilo; Page, Malcolm G P; Mourez, Michael; Weingart, Helge

    2014-01-01

    In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1-A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.

  14. Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk.

    PubMed

    Ishikawa, Toshihisa; Aw, Wanping; Kaneko, Kiyoko

    2013-11-04

    In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid) in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs), i.e., 421C>A (major) and 376C>T (minor), in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

  15. Marine Natural Products as Models to Circumvent Multidrug Resistance.

    PubMed

    Long, Solida; Sousa, Emília; Kijjoa, Anake; Pinto, Madalena M M

    2016-07-08

    Multidrug resistance (MDR) to anticancer drugs is a serious health problem that in many cases leads to cancer treatment failure. The ATP binding cassette (ABC) transporter P-glycoprotein (P-gp), which leads to premature efflux of drugs from cancer cells, is often responsible for MDR. On the other hand, a strategy to search for modulators from natural products to overcome MDR had been in place during the last decades. However, Nature limits the amount of some natural products, which has led to the development of synthetic strategies to increase their availability. This review summarizes the research findings on marine natural products and derivatives, mainly alkaloids, polyoxygenated sterols, polyketides, terpenoids, diketopiperazines, and peptides, with P-gp inhibitory activity highlighting the established structure-activity relationships. The synthetic pathways for the total synthesis of the most promising members and analogs are also presented. It is expected that the data gathered during the last decades concerning their synthesis and MDR-inhibiting activities will help medicinal chemists develop potential drug candidates using marine natural products as models which can deliver new ABC transporter inhibitor scaffolds.

  16. The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective.

    PubMed

    Sandhu, Padmani; Akhter, Yusuf

    2015-05-01

    The multidrug resistance has emerged as a major problem in the treatment of many of the infectious diseases. Tuberculosis (TB) is one of such disease caused by Mycobacterium tuberculosis. There is short term chemotherapy to treat the infection, but the main hurdle is the development of the resistance to antibiotics. This resistance is primarily due to the impermeable mycolic acid rich cell wall of the bacteria and other factors such as efflux of antibiotics from the bacterial cell. The MmpL (Mycobacterial Membrane Protein Large) proteins of mycobacteria are involved in the lipid transport and antibiotic efflux as indicated by the preliminary reports. We present here, comprehensive comparative sequence and structural analysis, which revealed topological signatures shared by the MmpL proteins and RND (Resistance Nodulation Division) multidrug efflux transporters. This provides evidence in support of the notion that they belong to the extended RND permeases superfamily. In silico modelled tertiary structures are in homology with an integral membrane component present in all of the RND efflux pumps. We document internal gene duplication and gene splitting events happened in the MmpL genes, which further elucidate the molecular functions of these putative transporters in an evolutionary perspective.

  17. Multiple ATP-binding cassette transporters are involved in insecticide resistance in the small brown planthopper, Laodelphax striatellus.

    PubMed

    Sun, H; Pu, J; Chen, F; Wang, J; Han, Z

    2017-03-16

    ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in the movement of various substrates, including drugs and insecticides, across the lipid membrane. Demonstration of the role of human ABC transporters in multidrug resistance has led to speculation that they might be an important mechanism controlling the fate of insecticides in insects. However, the role of ABC transporters in insects remains largely unknown. The small brown planthopper, Laodelphax striatellus Fallén, has developed resistance to most of the insecticides used for its control. Our goals were to identify the ABC transporters in La. striatellus and to examine their involvement in resistance mechanisms, using related strains resistant to chlorpyrifos, deltamethrin and imidacloprid, compared with the susceptible strain. Based on the transcriptome of La. striatellus, 40 full-length ABC transporters belonging to the ABCA-ABCH subfamilies were identified. Quantitative PCR revealed that over 20% of genes were significantly up-regulated in different resistant strains, and eight genes from the ABCB/C/D/G subfamilies were up-regulated in all three resistant strains, compared with the susceptible strain. Furthermore, synergism studies showed verapamil significantly enhanced insecticide toxicity in various resistant strains but not in the susceptible strain. These results suggest that ABC transporters might be involved in resistance to multiple insecticides in La. striatellus.

  18. ABC transporters and the proteasome complex are implicated in susceptibility to Stevens-Johnson syndrome and toxic epidermal necrolysis across multiple drugs.

    PubMed

    Nicoletti, Paola; Bansal, Mukesh; Lefebvre, Celine; Guarnieri, Paolo; Shen, Yufeng; Pe'er, Itsik; Califano, Andrea; Floratos, Aris

    2015-01-01

    Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) represent rare but serious adverse drug reactions (ADRs). Both are characterized by distinctive blistering lesions and significant mortality rates. While there is evidence for strong drug-specific genetic predisposition related to HLA alleles, recent genome wide association studies (GWAS) on European and Asian populations have failed to identify genetic susceptibility alleles that are common across multiple drugs. We hypothesize that this is a consequence of the low to moderate effect size of individual genetic risk factors. To test this hypothesis we developed Pointer, a new algorithm that assesses the aggregate effect of multiple low risk variants on a pathway using a gene set enrichment approach. A key advantage of our method is the capability to associate SNPs with genes by exploiting physical proximity as well as by using expression quantitative trait loci (eQTLs) that capture information about both cis- and trans-acting regulatory effects. We control for known bias-inducing aspects of enrichment based analyses, such as: 1) gene length, 2) gene set size, 3) presence of biologically related genes within the same linkage disequilibrium (LD) region, and, 4) genes shared among multiple gene sets. We applied this approach to publicly available SJS/TEN genome-wide genotype data and identified the ABC transporter and Proteasome pathways as potentially implicated in the genetic susceptibility of non-drug-specific SJS/TEN. We demonstrated that the innovative SNP-to-gene mapping phase of the method was essential in detecting the significant enrichment for those pathways. Analysis of an independent gene expression dataset provides supportive functional evidence for the involvement of Proteasome pathways in SJS/TEN cutaneous lesions. These results suggest that Pointer provides a useful framework for the integrative analysis of pharmacogenetic GWAS data, by increasing the power to detect aggregate effects

  19. Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

    PubMed

    Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

    2015-06-12

    The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance.

  20. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    PubMed Central

    Yanagisawa, Tatsuo; Kim, Kwang Sik; Yokoyama, Shigeyuki; Ohnishi, Makoto

    2015-01-01

    We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular l-glutamate via the GltT-GltM l-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the l-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC. PMID:26099588

  1. A Putative ABC Transporter Permease Is Necessary for Resistance to Acidified Nitrite and EDTA in Pseudomonas aeruginosa under Aerobic and Anaerobic Planktonic and Biofilm Conditions

    PubMed Central

    McDaniel, Cameron; Su, Shengchang; Panmanee, Warunya; Lau, Gee W.; Browne, Tristan; Cox, Kevin; Paul, Andrew T.; Ko, Seung-Hyun B.; Mortensen, Joel E.; Lam, Joseph S.; Muruve, Daniel A.; Hassett, Daniel J.

    2016-01-01

    Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite (A-NO2−, pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to A-NO2−. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to A-NO2−, but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with A-NO2− plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM A-NO2−, and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to A-NO2− in biofilms. A-NO2− sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, A-NO2− as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains. PMID:27064218

  2. 75 FR 49549 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-13

    ... Surface Transportation Board ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D), a noncarrier, has filed a verified notice of exemption... operation of this trackage in FD 35356, ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line...

  3. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

    SciTech Connect

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Bondar, Galyna; Zhu Quansheng; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira

    2010-04-15

    N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.

  4. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2).

    PubMed

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Scholz, Karoline; Bondar, Galyna; Zhu, Quansheng; Avliyakulov, Nuraly K; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira; Pushkin, Alexander

    2010-04-15

    N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.

  5. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

    PubMed Central

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Scholz, Karoline; Bondar, Galyna; Zhu, Quansheng; Avliyakulov, Nuraly K.; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira; Pushkin, Alexander

    2010-01-01

    N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC. PMID:20060011

  6. Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

    PubMed

    Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T; Zgurskaya, Helen I

    2015-10-01

    In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

  7. Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFα

    PubMed Central

    van der Heijden, J; de Jong, M C; Dijkmans, B; Lems, W; Oerlemans, R; Kathmann, I; Schalkwijk, C; Scheffer, G; Scheper, R; Jansen, G

    2004-01-01

    Objective: To determine whether overexpression of cell membrane associated drug efflux pumps belonging to the family of ATP binding cassette (ABC) proteins contributes to a diminished efficacy of sulfasalazine (SSZ) after prolonged cellular exposure to this disease modifying antirheumatic drug (DMARD). Methods: A model system of human T cells (CEM) was used to expose cells in vitro to increasing concentrations of SSZ for a period of six months. Cells were then characterised for the expression of drug efflux pumps: P-glycoprotein (Pgp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Results: Prolonged exposure of CEM cells to SSZ provoked resistance to SSZ as manifested by a 6.4-fold diminished antiproliferative effect of SSZ compared with parental CEM cells. CEM cells resistant to SSZ (CEM/SSZ) showed a marked induction of ABCG2/BCRP, Pgp expression was not detectable, while MRP1 expression was even down regulated. A functional role of ABCG2 in SSZ resistance was demonstrated by 60% reversal of SSZ resistance by the ABCG2 blocker Ko143. Release of the proinflammatory cytokine tumour necrosis factor α (TNFα) was threefold higher in CEM/SSZ cells than in CEM cells. Moreover, twofold higher concentrations of SSZ were required to inhibit TNFα release from CEM/SSZ cells compared with CEM cells. Conclusion: Collectively, ABCG2 induction, augmented TNFα release, and less efficient inhibition of TNFα production by SSZ may contribute to diminished efficacy after prolonged exposure to SSZ. These results warrant further clinical studies to verify whether drug efflux pumps, originally identified for their roles in cytostatic drug resistance, can also be induced by SSZ or other DMARDs. PMID:14722201

  8. Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette TransporterabcBA) Protects against Wild Type Brucella ovis in Rams

    PubMed Central

    Silva, Ana Patrícia C.; Macêdo, Auricélio A.; Costa, Luciana F.; Rocha, Cláudia E.; Garcia, Luize N. N.; Farias, Jade R. D.; Gomes, Priscilla P. R.; Teixeira, Gustavo C.; Fonseca, Kessler W. J.; Maia, Andréa R. F.; Neves, Gabriela G.; Romão, Everton L.; Silva, Teane M. A.; Mol, Juliana P. S.; Oliveira, Renata M.; Araújo, Márcio S. S.; Nascimento, Ernane F.; Martins-Filho, Olindo A.; Brandão, Humberto M.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams. PMID:26317399

  9. Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein.

    PubMed

    Matsushima, Soichiro; Maeda, Kazuya; Kondo, Chihiro; Hirano, Masaru; Sasaki, Makoto; Suzuki, Hiroshi; Sugiyama, Yuichi

    2005-09-01

    Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17beta-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.

  10. 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

  11. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system.

    PubMed

    Szczepanowski, Rafael; Krahn, Irene; Linke, Burkhard; Goesmann, Alexander; Pühler, Alfred; Schlüter, Andreas

    2004-11-01

    Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP

  12. MFS transporters required for multidrug/multixenobiotic (MD/MX) resistance in the model yeast: understanding their physiological function through post-genomic approaches

    PubMed Central

    dos Santos, Sandra C.; Teixeira, Miguel C.; Dias, Paulo J.; Sá-Correia, Isabel

    2014-01-01

    Multidrug/Multixenobiotic resistance (MDR/MXR) is a widespread phenomenon with clinical, agricultural and biotechnological implications, where MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a key role in the acquisition of resistance. However, although these proteins have been traditionally considered drug exporters, the physiological function of MDR/MXR transporters and the exact mechanism of their involvement in resistance to cytotoxic compounds are still open to debate. In fact, the wide range of structurally and functionally unrelated substrates that these transporters are presumably able to export has puzzled researchers for years. The discussion has now shifted toward the possibility of at least some MDR/MXR transporters exerting their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds, while the hypothesis that MDR/MXR transporters may have evolved in nature for other purposes than conferring chemoprotection has been gaining momentum in recent years. This review focuses on the drug transporters of the Major Facilitator Superfamily (MFS; drug:H+ antiporters) in the model yeast Saccharomyces cerevisiae. New insights into the natural roles of these transporters are described and discussed, focusing on the knowledge obtained or suggested by post-genomic research. The new information reviewed here provides clues into the unexpectedly complex roles of these transporters, including a proposed indirect regulation of the stress response machinery and control of membrane potential and/or internal pH, with a special emphasis on a genome-wide view of the regulation and evolution of MDR/MXR-MFS transporters. PMID:24847282

  13. Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy.

    PubMed

    Luna-Tortós, Carlos; Fedrowitz, Maren; Löscher, Wolfgang

    2010-06-01

    Resistance to antiepileptic drugs (AEDs) is one of the most serious problems in the treatment of epilepsy. Accumulating experimental evidence suggests that increased expression of the drug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier may be involved in the mechanisms leading to AED resistance. In addition to Pgp, increased expression of several multidrug resistance-associated proteins (MRPs) has been determined in epileptogenic brain regions of patients with pharmacoresistant epilepsy. However, it is not known whether AEDs are substrates for MRPs. In the present experiments, we evaluated whether common AEDs are transported by human MRPs (MRP1, 2 and 5) that are overexpressed in AED resistant epilepsy. For this purpose, we used a highly sensitive assay (concentration equilibrium transport assay; CETA) in polarized kidney cell lines (LLC, MDCKII) transfected with human MRPs. The assay was validated by known MRP substrates, including calcein-AM (MRP1), vinblastine (MRP2) and chloromethylfluorescein diacetate (CMFDA; MRP5). The directional transport determined with these drugs in MRP-transfected cell lines could be blocked with the MRP inhibitor MK571. However, in contrast to transport of known MRP substrates, none of the common AEDs (carbamazepine, valproate, levetiracetam, phenytoin, lamotrigine and phenobarbital) used in this study was transported by MRP1, MRP2 or MRP5. A basolateral-to-apical transport of valproate, which could be inhibited by MK571 and probenecid, was determined in LLC cells (both wildtype and transfected), but the specific transporter involved was not identified. The data indicate that common AEDs are not substrates for human MRP1, MRP2 or MRP5, at least in the in vitro models used in this study.

  14. The Deviant ATP-binding Site of the Multidrug Efflux Pump Pdr5 Plays an Active Role in the Transport Cycle*

    PubMed Central

    Furman, Christopher; Mehla, Jitender; Ananthaswamy, Neeti; Arya, Nidhi; Kulesh, Bridget; Kovach, Ildiko; Ambudkar, Suresh V.; Golin, John

    2013-01-01

    Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites. PMID:24019526

  15. N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

    PubMed

    Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C

    2016-01-22

    Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity.

  16. Polymorphism of the Plasmodium falciparum multidrug resistance and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon.

    PubMed

    Huaman, Maria Cecilia; Roncal, Norma; Nakazawa, Shusuke; Long, Ton That Ai; Gerena, Lucia; Garcia, Coralith; Solari, Lely; Magill, Alan J; Kanbara, Hiroji

    2004-05-01

    In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P. falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance. The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.

  17. Ospdr9, which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots.

    PubMed

    Moons, Ann

    2003-10-23

    Little is known about the role of pleiotropic drug resistance (PDR)-type ATP-binding (ABC) proteins in plant responses to environmental stresses. We characterised ospdr9, which encodes a rice ABC protein with a reverse (ABC-TMS(6))(2) configuration. Polyethylene glycol and the heavy metals Cd (20 microM) and Zn (30 microM) rapidly and markedly induced ospdr9 in roots of rice seedlings. Hypoxic stress also induced ospdr9 in rice roots, salt stress induced ospdr9 at low levels but cold and heat shock had no effect. The plant growth regulator jasmonic acid, the auxin alpha-naphthalene acetic acid and the cytokinin 6-benzylaminopurine triggered ospdr9 expression. The antioxidants dithiothreitol and ascorbic acid rapidly and markedly induced ospdr9 in rice roots; the strong oxidant hydrogen peroxide also induced ospdr9 but at three times lower levels. The results suggested that redox changes may be involved in the abiotic stress response regulation of ospdr9 in rice roots.

  18. 75 FR 11991 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF TRANSPORTATION Surface Transportation Board ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D), a noncarrier, has filed a verified notice of...

  19. Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice.

    PubMed

    Riquelme-Neira, Roberto; Retamal-Díaz, Angello; Acuña, Francisca; Riquelme, Pablo; Rivera, Alejandra; Sáez, Darwin; Oñate, Angel

    2013-08-12

    The immunogenicity of a DNA vaccine containing an open reading frame (ORF) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the open reading frame with homology to an ABC-type transporter (pV278a) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pV278a had a dominant immunoglobulin G2a (IgG2a) response. This DNA vaccine elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 278a protein. Upon stimulation with an appropriate recombinant protein or crude Brucella protein, the vaccine did not induce IL-4, suggesting a typical T-helper (TH1) response. Furthermore, the vaccine induced protection in BALB/c mice when challenged with the virulent strain Brucella abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery of the homologous of an ABC-type transporter antigen, and provides the first evidence of a protective effect of this antigen in the construction of vaccines against B. abortus.

  20. Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR.

    PubMed

    Schumacher, Maria A; Brennan, Richard G

    2003-03-01

    Multidrug transporters and their transcriptional regulators are key components of bacterial multidrug resistance (MDR). How these multidrug binding proteins can recognize such chemically disparate compounds represents a fascinating question from a structural standpoint and an important question in future drug development efforts. The Staphylococcus aureus multidrug binding regulator, QacR, is soluble and recognizes an especially wide range of structurally dissimilar compounds, properties making it an ideal model system for deciphering the molecular basis of multidrug recognition. Recent structures of QacR have afforded the first view of any MDR protein bound to multiple drugs, revealing key structural features of multidrug recognition, including a multisite binding pocket.

  1. [The role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria].

    PubMed

    Hasdemir, Ufuk

    2007-04-01

    Multiple antibiotic resistance of clinically important bacteria are of major concern worldwide. Alterations of drug targets or enzymatic inactivation of antimicrobial agents are the well known mechanisms of antimicrobial drug resistance. Besides these well known mechanisms, recent studies have shown that a further resistance mechanism, active drug efflux, has become increasingly important in the current threat of multidrug resistance. It involves certain bacterial transport proteins which pump out toxic antimicrobial compounds from the cell. Drug efflux pump proteins in bacteria fall into five distinct protein super families [ATP binding cassette super family (ABC), Major facilitator super family (MFS), Small multidrug resistance super family (SMR), Multidrug and toxic compound extrusion (MATE) super family, Resistance-nodulation-cell division (RND) super family] and are mostly encoded by chromosomal genes. Among them, the members of RND protein super family are widely distrubuted in Gram negative bacteria and play siginificant role in both, intrinsic and acquired multidrug resistance of these bacteria with very wide substrate specificity. RND type multidrug efflux proteins usually function together with an outer membrane canal protein (OMP) and a membrane fusion protein (MFP) to pump out drugs. AcrAB-TolC of Escherichia coli and MexAB-OprM of Pseudomonas aeruginosa are the typical examples of these tripartite systems. They are constitutively expressed in wild type cells and play significant role in intrinsic resistance of these bacteria. However, multidrug resistance which is of major clinical significance, rises as a result of overexpression of these pump systems due to mutations and elevated levels of resistance are recorded to structurally unrelated antimicrobial drugs such as fluoroquinolones, beta-lactams, tetracyclines, chloramphenicol, trimethoprim, aminoglycosides and toxic compunds. Synthesis of RND type pump proteins are regulated by complex genetic

  2. Involvement of the Putative ATP-Dependent Efflux Proteins PatA and PatB in Fluoroquinolone Resistance of a Multidrug-Resistant Mutant of Streptococcus pneumoniae

    PubMed Central

    Marrer, Estelle; Schad, Karen; Satoh, Andreas T.; Page, Malcolm G. P.; Johnson, Maggie M.; Piddock, Laura J. V.

    2006-01-01

    The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains. PMID:16436727

  3. Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5

    PubMed Central

    Sauna, Zuben E.; Bohn, Sherry Supernavage; Rutledge, Robert; Dougherty, Michael P.; Cronin, Susan; May, Leopold; Xia, Di; Ambudkar, Suresh V.; Golin, John

    2008-01-01

    The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable Km(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport. PMID:18842589

  4. Gene expression profiling of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR.

    PubMed

    Toselli, Francesca; de Waziers, Isabelle; Dutheil, Mary; Vincent, Marc; Wilce, Peter A; Dodd, Peter R; Beaune, Philippe; Loriot, Marie-Anne; Gillam, Elizabeth M J

    2015-01-01

    1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

  5. ABC's of Being Smart

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    Determining what giftedness is all about means focusing on many aspects of the individual. In this paper, the author focuses on letter D of the ABC's of being smart. She starts with specifics about giftedness (details), and then moves on to some ways of thinking (dispositions).

  6. 1968 ABC Summer Program.

    ERIC Educational Resources Information Center

    Kerr, Frances M.; Russell, Valerie E.

    A talent development project at Mount Holyoke College, part of A Better Chance (ABC)-Independent Schools Talent Search program, was offered during the summer of 1968 to 71 disadvantaged high school students from 13 states. Major aims of the program were to help these students with college potential to strengthen their academic skills and…

  7. Involvement of the Leaf-Specific Multidrug and Toxic Compound Extrusion (MATE) Transporter Nt-JAT2 in Vacuolar Sequestration of Nicotine in Nicotiana tabacum

    PubMed Central

    Shitan, Nobukazu; Minami, Shota; Morita, Masahiko; Hayashida, Minaho; Ito, Shingo; Takanashi, Kojiro; Omote, Hiroshi; Moriyama, Yoshinori; Sugiyama, Akifumi; Goossens, Alain; Moriyasu, Masataka; Yazaki, Kazufumi

    2014-01-01

    Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues. PMID:25268729

  8. Modulation of human multidrug resistance protein (MRP) 1 (ABCC1) and MRP2 (ABCC2) transport activities by endogenous and exogenous glutathione-conjugated catechol metabolites.

    PubMed

    Slot, Andrew J; Wise, Dana D; Deeley, Roger G; Monks, Terrence J; Cole, Susan P C

    2008-03-01

    Members of the multidrug resistance protein (MRP/ABCC) subfamily of ATP-binding cassette proteins transport a wide array of anionic compounds, including sulfate, glucuronide, and glutathione (GSH) conjugates. The present study tested the ATP-dependent vesicular transport of leukotriene C(4) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) mediated by the MRP1 and MRP2 transporters in the presence of six potential modulators from three different classes of GSH-conjugated catechol metabolites: the ecstasy metabolite 5-(glutathion-S-yl)-N-methyl-alpha-methyldopamine (5-GS-N-Me-alpha-MeDA), the caffeic acid metabolite 2-(glutathion-S-yl)-caffeic acid (2-GS-CA), and four GSH conjugates of 2-hydroxy (OH) and 4-OH estrogens (GS estrogens). MRP1-mediated E(2)17betaG transport was inhibited in a competitive manner with a relative order of potency of GS estrogens (IC(50) <1 microM) > 2-GS-CA (IC(50) 3 microM) > 5-GS-N-Me-alpha-MeDA (IC(50) 31 microM). MRP2-mediated transport was inhibited with a similar order of potency, except the 2-hydroxy-4-(glutathion-S-yl)-estradiol and 4-hydroxy-2-(glutathion-S-yl)-estradiol conjugates were approximately 50- and 300-fold less potent, respectively. Transport activity was unaffected by N-acetylcysteine conjugates of N-Me-alpha-MeDA and CA. The position of GSH conjugation appears important as all four GS estrogen conjugates tested were potent inhibitors of MRP1 transport, but only the 2-hydroxy-1-(glutathion-S-yl)-estradiol and 2-hydroxy-1-(glutathion-S-yl)-estrone conjugates were potent inhibitors of MRP2-mediated transport. In conclusion, we have identified three new classes of MRP1 and MRP2 modulators and demonstrated that one of these, the estrogen conjugates, shows unanticipated differences in their interactions with the two transporters.

  9. Elevated expression of Nrf2 mediates multidrug resistance in CD133+ head and neck squamous cell carcinoma stem cells

    PubMed Central

    Lu, Bao-Cai; Li, Jing; Yu, Wen-Fa; Zhang, Guo-Zheng; Wang, Hui-Min; Ma, Hui-Min

    2016-01-01

    Enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC sub-family G member 2 (ABCG2) in cancer stem cells (CSCs) plays a major role in chemotherapeutic drug efflux, which results in therapy failure and tumor relapse. In addition to downregulating apoptosis in CSCs, it has been reported that the transcriptional upregulation of the redox sensing factor Nrf2 is involved in the upregulation of ABCG2 expression and consequent chemoresistance. The current study investigated the presence of cancer stem-like side population (SP) cells from head and neck squamous cell carcinoma (HNSCC) samples, and evaluated the Nrf2 expression profile and multidrug resistance properties of HNSCC stem cells. Fluorescence-activated cell sorting was used for SP cells detection, while reverse transcription-polymerase chain reaction was used for the analysis of Nrf2 expression. The present study identified ~2.1% SP cells present in HNSCC specimens, which were positive for cluster of differentiation (CD)133 expression and displayed significantly elevated messenger RNA expression of Nrf2, compared with non-SP cells. These data suggest that the ABC transporter ABCG2 is highly upregulated in SP cells, and this results in multidrug resistance. In addition, these CD133+ cells underwent rapid proliferation and exhibited high self-renewal and tumorigenic properties. Taken together, the present findings suggest that elevated expression of Nrf2 mediated drug resistance in HNSCC CSCs, which may be one of the causative factors for cancer treatment failure. Therefore, novel anti-cancer drugs that downregulate the Nrf2 signaling pathway could effectively improve the treatment and survival rate of patients with HNSCC. PMID:28101198

  10. Multidrug resistance in chronic myeloid leukaemia: how much can we learn from MDR–CML cell lines?

    PubMed Central

    Rumjanek, Vivian M.; Vidal, Raphael S.; Maia, Raquel C.

    2013-01-01

    The hallmark of CML (chronic myeloid leukaemia) is the BCR (breakpoint cluster region)–ABL fusion gene. CML evolves through three phases, based on both clinical and pathological features: a chronic phase, an accelerated phase and blast crisis. TKI (tyrosine kinase inhibitors) are the treatment modality for patients with chronic phase CML. The therapeutic potential of the TKI imatinib is affected by BCR–ABL dependent an independent mechanisms. Development of MDR (multidrug resistance) contributes to the overall clinical resistance. MDR involves overexpression of ABC -transporters (ATP-binding-cassette transporter) among other features. MDR studies include the analysis of cancer cell lines selected for resistance. CML blast crisis is accompanied by increased resistance to apoptosis. This work reviews the role played by the influx transporter OCT1 (organic cation transporter 1), by efflux ABC transporters, molecules involved in the modulation of apoptosis (p53, Bcl-2 family, CD95, IAPs (inhibitors of apoptosis protein)], Hh and Wnt/β-catenin pathways, cytoskeleton abnormalities and other features described in leukaemic cells of clinical samples and CML cell lines. An MDR cell line, Lucena-1, generated from K562 by stepwise exposure to vincristine, was used as our model and some potential anticancer drugs effective against the MDR cell line and patients’ samples are presented. PMID:24070327

  11. Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine.

    PubMed

    Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Staud, Frantisek

    2016-01-01

    Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer.

  12. High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

    PubMed Central

    Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

    2001-01-01

    In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

  13. Nanodrug delivery in reversing multidrug resistance in cancer cells

    PubMed Central

    Kapse-Mistry, Sonali; Govender, Thirumala; Srivastava, Rohit; Yergeri, Mayur

    2014-01-01

    Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins (MRP1, MRP2), and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1α gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-κB. “Theragnostics” combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome

  14. Moving out: from sterol transport to drug resistance - the ABCG subfamily of efflux pumps.

    PubMed

    Moitra, Karobi; Silverton, Latoya; Limpert, Katy; Im, Kate; Dean, Michael

    2011-01-01

    The ATP binding cassette (ABC) proteins are typically ATP-driven transmembrane pumps that have been evolutionarily conserved from bacteria to humans. In humans these transporters are subdivided into seven subfamilies, ranging from A to G. The ABCG subfamily of transporters is the primary focus of this review. This subfamily of proteins has been conserved throughout evolution and plays a central role in several cellular processes, such as sterol homeostasis and multidrug resistance. Functional polymorphisms/mutations in some of these G-subfamily transporters have clinical consequences in humans.

  15. Fallopia japonica, a Natural Modulator, Can Overcome Multidrug Resistance in Cancer Cells

    PubMed Central

    Eid, Safaa Yehia; El-Readi, Mahmoud Zaki; Ashour, Mohamed Lotfy; Wink, Michael

    2015-01-01

    Resistance of cancer cells to chemotherapy is controlled by the decrease of intracellular drug accumulation, increase of detoxification, and diminished propensity of cancer cells to undergo apoptosis. ATP-binding cassette (ABC) membrane transporters with intracellular metabolic enzymes contribute to the complex and unresolved phenomenon of multidrug resistance (MDR). Natural products as alternative medicine have great potential to discover new MDR inhibitors with diverse modes of action. In this study, we characterized several extracts of traditional Chinese medicine (TCM) plants (N = 16) for their interaction with ABC transporters, cytochrome P3A4 (CYP3A4), and glutathione-S-transferase (GST) activities and their cytotoxic effect on different cancer cell lines. Fallopia japonica (FJ) (Polygonaceae) shows potent inhibitory effect on CYP3A4 P-glycoprotein activity about 1.8-fold when compared to verapamil as positive control. FJ shows significant inhibitory effect (39.81%) compared with the known inhibitor ketoconazole and 100 μg/mL inhibited GST activity to 14 μmol/min/mL. FJ shows moderate cytotoxicity in human Caco-2, HepG-2, and HeLa cell lines; IC50 values were 630.98, 198.80, and 317.37 µg/mL, respectively. LC-ESI-MS were used to identify and quantify the most abundant compounds, emodin, polydatin, and resveratrol, in the most active extract of FJ. Here, we present the prospect of using Fallopia japonica as natural products to modulate the function of ABC drug transporters. We are conducting future study to evaluate the ability of the major active secondary metabolites of Fallopia japonica to modulate MDR and their impact in case of failure of chemotherapy. PMID:26346937

  16. Chalcogenopyrylium Dyes as Differential Modulators of Organic Anion Transport by Multidrug Resistance Protein 1 (MRP1), MRP2, and MRP4

    PubMed Central

    Myette, Robert L.; Conseil, Gwenaëlle; Ebert, Sean P.; Wetzel, Bryan; Detty, Michael R.

    2013-01-01

    Multidrug resistance proteins (MRPs) mediate the ATP-dependent efflux of structurally diverse compounds, including anticancer drugs and physiologic organic anions. Five classes of chalcogenopyrylium dyes (CGPs) were examined for their ability to modulate transport of [3H]estradiol glucuronide (E217βG; a prototypical MRP substrate) into MRP-enriched inside-out membrane vesicles. Additionally, some CGPs were tested in intact transfected cells using a calcein efflux assay. Sixteen of 34 CGPs inhibited MRP1-mediated E217βG uptake by >50% (IC50 values: 0.7–7.6 µM). Of 9 CGPs with IC50 values ≤2 µM, two belonged to class I, two to class III, and five to class V. When tested in the intact cells, only 4 of 16 CGPs (at 10 µM) inhibited MRP1-mediated calcein efflux by >50% (III-1, V-3, V-4, V-6), whereas a fifth (I-5) inhibited efflux by just 23%. These five CGPs also inhibited [3H]E217βG uptake by MRP4. In contrast, their effects on MRP2 varied, with two (V-4, V-6) inhibiting E217βG transport (IC50 values: 2.0 and 9.2 µM) and two (V-3, III-1) stimulating transport (>2-fold), whereas CGP I-5 had no effect. Strikingly, although V-3 and V-4 had opposite effects on MRP2 activity, they are structurally identical except for their chalcogen atom (Se versus Te). This study is the first to identify class V CGPs, with their distinctive methine or trimethine linkage between two disubstituted pyrylium moieties, as a particularly potent class of MRP modulators, and to show that, within this core structure, differences in the electronegativity associated with a chalcogen atom can be the sole determinant of whether a compound will stimulate or inhibit MRP2. PMID:23530018

  17. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  18. Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

    PubMed Central

    Tedesco, Pietro; Visone, Marco; Parrilli, Ermenegilda; Tutino, Maria Luisa; Perrin, Elena; Maida, Isabel; Fani, Renato; Ballestriero, Francesco; Santos, Radleigh; Pinilla, Clemencia; Di Schiavi, Elia; Tegos, George; de Pascale, Donatella

    2015-01-01

    This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms. PMID:26587842

  19. Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model.

    PubMed

    Tedesco, Pietro; Visone, Marco; Parrilli, Ermenegilda; Tutino, Maria Luisa; Perrin, Elena; Maida, Isabel; Fani, Renato; Ballestriero, Francesco; Santos, Radleigh; Pinilla, Clemencia; Di Schiavi, Elia; Tegos, George; de Pascale, Donatella

    2015-01-01

    This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms' intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.

  20. Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells.

    PubMed

    Sun, Hua; Wang, Xiao; Zhou, Xiaotong; Lu, Danyi; Ma, Zhiguo; Wu, Baojian

    2015-10-01

    Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-3'-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

  1. Polymorphisms in the human organic cation transporter and the multidrug resistance gene: correlation with imatinib levels and clinical course in patients with chronic myeloid leukemia.

    PubMed

    Vine, Jacob; Cohen, Sara Bar; Ruchlemer, Rosa; Goldschmidt, Neta; Levin, Moshe; Libster, Diana; Gural, Alexander; Gatt, Moshe E; Lavie, David; Ben-Yehuda, Dina; Rund, Deborah

    2014-11-01

    The optimal tyrosine kinase inhibitor for any individual patient with chronic myeloid leukemia (CML) is not predictable. Pharmacogenetic parameters and trough levels of imatinib (IM) have each been independently correlated with response. We therefore studied the human organic cation transporter (hOCT1) and multidrug resistance (MDR1) single nucleotide polymorphisms (SNPs) and correlated these with IM levels and major molecular response (MMR) (3-log reduction) in 84 patients with CML, the first such study performed in Caucasians. We studied MDR1 G2677T and C3435T, and for hOCT1, C480G and A1222G. IM levels varied significantly with dose (< or > 400 mg/day) (p = 0.038) and were significantly lower in 20 patients who lost MMR (p = 0.042). Adjusting for dose, trough IM levels were not significantly correlated with SNPs. Patients with MDR1 3435 TT had significantly longer times to MMR compared to CC/CT genotypes (p = 0.047). Genotypes did not predict treatment failure when controlling for IM levels. We conclude that IM levels, but not the SNPs studied here, determine IM failure.

  2. Organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the placenta and fetal tissues: expression profile and fetus protective role at different stages of gestation.

    PubMed

    Ahmadimoghaddam, Davoud; Zemankova, Lenka; Nachtigal, Petr; Dolezelova, Eva; Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Kacerovský, Marian; Micuda, Stanislav; Staud, Frantisek

    2013-03-01

    In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP(+)), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP(+) reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP(+) decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP(+) from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.

  3. 3D-QSAR AND CONTOUR MAP ANALYSIS OF TARIQUIDAR ANALOGUES AS MULTIDRUG RESISTANCE PROTEIN-1 (MRP1) INHIBITORS

    PubMed Central

    Kakarla, Prathusha; Inupakutika, Madhuri; Devireddy, Amith R.; Gunda, Shravan Kumar; Willmon, Thomas Mark; Ranjana, KC; Shrestha, Ugina; Ranaweera, Indrika; Hernandez, Alberto J.; Barr, Sharla; Varela, Manuel F.

    2016-01-01

    One of the major obstacles to the successful chemotherapy towards several cancers is multidrug resistance of human cancer cells to anti-cancer drugs. An important contributor to multidrug resistance is the human multidrug resistance protein-1 transporter (MRP1), which is an efflux pump of the ABC (ATP binding cassette) superfamily. Thus, highly efficacious, third generation MRP1 inhibitors, like tariquidar analogues, are promising inhibitors of multidrug resistance and are under clinical trials. To maximize the efficacy of MRP1 inhibitors and to reduce systemic toxicity, it is important to limit the exposure of MRP1 inhibitors and anticancer drugs to normal tissues and to increase their co-localization with tumor cells. Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) associated with 3D-Quantitiative structure-activity relationship (3D-QSAR) studies were performed on a series of tariquidar analogues, as selective MDR modulators. Best predictability was obtained with CoMFA model r2(non-cross-validated square of correlation coefficient) = 0.968, F value = 151.768 with five components, standard error of estimate = 0.107 while the CoMSIA yielded r2 = 0.982, F value = 60.628 with six components, and standard error of estimate = 0.154. These results indicate that steric, electrostatic, hydrophobic (lipophilic), and hydrogen bond donor substituents play significant roles in multidrug resistance modulation of tariquidar analogues upon MRP1. The tariquidar analogue and MRP1 binding and stability data generated from CoMFA and CoMSIA based 3D–contour maps may further aid in study and design of tariquidar analogues as novel, potent and selective MDR modulator drug candidates. PMID:26913287

  4. The role of multidrug resistance protein (MRP-1) as an active efflux transporter on blood-brain barrier (BBB) permeability.

    PubMed

    Lingineni, Karthik; Belekar, Vilas; Tangadpalliwar, Sujit R; Garg, Prabha

    2017-01-03

    Drugs acting on central nervous system (CNS) may take longer duration to reach the market as these compounds have a higher attrition rate in clinical trials due to the complexity of the brain, side effects, and poor blood-brain barrier (BBB) permeability compared to non-CNS-acting compounds. The roles of active efflux transporters with BBB are still unclear. The aim of the present work was to develop a predictive model for BBB permeability that includes the MRP-1 transporter, which is considered as an active efflux transporter. A support vector machine model was developed for the classification of MRP-1 substrates and non-substrates, which was validated with an external data set and Y-randomization method. An artificial neural network model has been developed to evaluate the role of MRP-1 on BBB permeation. A total of nine descriptors were selected, which included molecular weight, topological polar surface area, ClogP, number of hydrogen bond donors, number of hydrogen bond acceptors, number of rotatable bonds, P-gp, BCRP, and MRP-1 substrate probabilities for model development. We identified 5 molecules that fulfilled all criteria required for passive permeation of BBB, but they all have a low logBB value, which suggested that the molecules were effluxed by the MRP-1 transporter.

  5. Risk Factors for Development of Cholestatic Drug-Induced Liver Injury: Inhibition of Hepatic Basolateral Bile Acid Transporters Multidrug Resistance-Associated Proteins 3 and 4

    PubMed Central

    Köck, Kathleen; Ferslew, Brian C.; Netterberg, Ida; Yang, Kyunghee; Urban, Thomas J.; Swaan, Peter W.; Stewart, Paul W.

    2014-01-01

    Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 μM) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e.g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. PMID:24154606

  6. 3D structure of AcrB: the archetypal multidrug efflux transporter of Escherichia coli likely captures substrates from periplasm.

    PubMed

    Elkins, Christopher A; Nikaido, Hiroshi

    2003-02-01

    Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and its homologs are the major mechanisms that give most Gram-negative bacteria their characteristic intrinsic resistance to a variety of lipophilic drugs, dyes, and detergents. Most recently, the structure of the transporter AcrB was elucidated at high resolution [Nature 419(2002)587]. It is a particularly significant achievement since integral membrane proteins are notoriously elusive structures for crystallography. The striking features of this trimeric pump, such as the presence of potential substrate-binding sites in the periplasmic domain and the possibility of direct interaction with the end of TolC tunnel, refine our understanding of the mode of action of this tripartite efflux transport complex.

  7. Multidrug Resistance: Physiological Principles and Nanomedical Solutions

    PubMed Central

    Storm, Gert; Kiessling, Fabian; Lammers, Twan

    2014-01-01

    Multidrug (MDR) resistance is a pathophysiological phenomenon employed by cancer cells which limits the prolonged and effective use of chemotherapeutic agents. MDR is primarily based on the over-expression of drug efflux pumps in the cellular membrane. Prominent examples of such efflux pumps, which belong to the ATP-binding cassette (ABC) superfamily of proteins, are Pgp (P-glycoprotein) and MRP (multidrug resistance-associated protein), nowadays officially known as ABCB1 and ABCC1. Over the years, several strategies have been evaluated to overcome MDR, based not only on the use of low-molecular-weight MDR modulators, but also on the implementation of 1-100(0) nm-sized drug delivery systems. In the present manuscript, after introducing the most important physiological principles of MDR, we summarize prototypic nanomedical strategies to overcome multidrug resistance, including the use of carrier materials with intrinsic anti-MDR properties, the use of nanomedicines to modify the mode of cellular uptake, and the co-formulation of chemotherapeutic drugs together with low- and high-molecular-weight MDR inhibitors within a single drug delivery system. While certain challenges still need to be overcome before such constructs and concepts can be widely applied in the clinic, the insights obtained and the progress made strongly suggest that nanomedicine formulations hold significant potential for improving the treatment of multidrug-resistant malignancies. PMID:24120954

  8. Roles of P-glycoprotein and multidrug resistance protein in transporting para-aminosalicylic acid and its N-acetylated metabolite in mice brain

    PubMed Central

    Hong, Lan; Xu, Cong; O'Neal, Stefanie; Bi, Hui-chang; Huang, Min; Zheng, Wei; Zeng, Su

    2014-01-01

    Aim: Para-aminosalicylic acid (PAS) is effective in the treatment of manganism-induced neurotoxicity (manganism). In this study we investigated the roles of P-glycoprotein (MDR1a) and multidrug resistance protein (MRP) in transporting PAS and its N-acetylated metabolite AcPAS through blood-brain barrier. Methods: MDR1a-null or wild-type mice were intravenously injected with PAS (200 mg/kg). Thirty minutes after the injection, blood samples and brains were collected, and the concentrations of PAS and AcPAS in brain capillaries and parenchyma were measured using HPLC. Both MDCK-MDR1 and MDCK-MRP1 cells that overexpressed P-gp and MRP1, respectively, were used in two-chamber Transwell transport studies in vitro. Results: After injection of PAS, the brain concentration of PAS was substantially higher in MDR1a-null mice than in wild-type mice, but the brain concentration of AcPAS had no significant difference between MDR1a-null mice and wild-type mice. Concomitant injection of PAS with the MRP-specific inhibitor MK-571 (50 mg/kg) further increased the brain concentration of PAS in MDR1a-null mice, and increased the brain concentration of AcPAS in both MDR1a-null mice and wild-type mice. Two-chamber Transwell studies with MDCK-MDR1 cells demonstrated that PAS was not only a substrate but also a competitive inhibitor of P-gp, while AcPAS was not a substrate of P-gp. Two-chamber Transwell studies with the MDCK-MRP1 cells showed that MRP1 had the ability to transport both PAS and AcPAS across the BBB. Conclusion: P-gp plays a major role in the efflux of PAS from brain parenchyma into blood in mice, while MRP1 is involved in both PAS and AcPAS transport in the brain. PMID:25418377

  9. Learning the ABC of oral fungal drug resistance.

    PubMed

    Cannon, R D; Holmes, A R

    2015-12-01

    ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.

  10. The synthesis and biodistribution of [(11)C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo.

    PubMed

    Hume, W Ewan; Shingaki, Tomotaka; Takashima, Tadayuki; Hashizume, Yoshinobu; Okauchi, Takashi; Katayama, Yumiko; Hayashinaka, Emi; Wada, Yasuhiro; Kusuhara, Hiroyuki; Sugiyama, Yuichi; Watanabe, Yasuyoshi

    2013-12-15

    In order to develop a new positron emission tomography (PET) probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1), (11)C-labelled metformin was synthesized and then evaluated as a PET probe. [(11)C]Metformin ([(11)C]4) was synthesized in three steps, from [(11)C]methyl iodide. Evaluation by small animal PET of [(11)C]4 showed that there was increased concentrations of [(11)C]4 in the livers of mice pre-treated with pyrimethamine, a potential inhibitor of MATEs, inhibiting the hepatobiliary excretion of metformin. Radiometabolite analysis showed that [(11)C]4 was not degraded in vivo during the PET scan. Biodistribution studies were undertaken and the organ distributions were extrapolated into a standard human model. In conclusion, [(11)C]4 may be useful as a PET probe to non-invasively study the in vivo function of hepatobiliary transport and drug-drug interactions, mediated by MATE1 in future clinical investigations.

  11. Reporter Dyes Demonstrate Functional Expression of Multidrug Resistance Proteins in the Marine Flatworm Macrostomum lignano: The Sponge-Derived Dye Ageladine A Is Not a Substrate of These Transporters

    PubMed Central

    Tietje, Kristin; Rivera-Ingraham, Georgina; Petters, Charlotte; Abele, Doris; Dringen, Ralf; Bickmeyer, Ulf

    2013-01-01

    The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy. PMID:24135911

  12. Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP

    PubMed Central

    Nöll, Anne; Thomas, Christoph; Herbring, Valentina; Zollmann, Tina; Barth, Katja; Mehdipour, Ahmad Reza; Tomasiak, Thomas M.; Brüchert, Stefan; Joseph, Benesh; Abele, Rupert; Oliéric, Vincent; Wang, Meitian; Diederichs, Kay; Stroud, Robert M.; Pos, Klaas M.; Tampé, Robert

    2017-01-01

    ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetri