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

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Uncaria alkaloids reverse ABCB1-mediated cancer multidrug resistance

PubMed Central

Huang, Bao-Yuan; Zeng, Yu; Li, Ying-Jie; Huang, Xiao-Jun; Hu, Nan; Yao, Nan; Chen, Min-Feng; Yang, Zai-Gang; Chen, Zhe-Sheng; Zhang, Dong-Mei; Zeng, Chang-Qing

2017-01-01

The overexpression of ATP-binding cassette (ABC) transporters is the main cause of cancer multidrug resistance (MDR), which leads to chemotherapy failure. Uncaria alkaloids are the major active components isolated from uncaria, which is a common Chinese herbal medicine. In this study, the MDR-reversal activities of uncaria alkaloids, including rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine (Icory), hirsutine and hirsuteine, were screened; they all exhibited potent reversal efficacy when combined with doxorubicin. Among them, Icory significantly sensitized ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells to vincristine, doxorubicin and paclitaxel, but not to the non-ABCB1 substrate cisplatin. Noteworthy, Icory selectively reversed ABCB1-overexpressing MDR cancer cells but not ABCC1- or ABCG2-mediated MDR. Further mechanistic study revealed that Icory increased the intracellular accumulation of doxorubicin in ABCB1-overexpressing cells by blocking the efflux function of ABCB1. Instead of inhibiting ABCB1 expression and localization, Icory acts as a substrate of the ABCB1 transporter by competitively binding to substrate binding sites. Collectively, these results indicated that Icory reversed ABCB1-mediated MDR by suppressing its efflux function, and it would be beneficial to increase the efficacy of these types of uncaria alkaloids and develop them to be selective ABCB1-mediated MDR-reversal agents. PMID:28534954

Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains.

PubMed

Hinrichs, John W J; Klappe, Karin; van Riezen, Manon; Kok, Jan W

2005-11-01

We have recently shown that two ATP binding cassette (ABC) transporters are enriched in Lubrol-resistant noncaveolar membrane domains in multidrug-resistant human cancer cells [Hinrichs, J. W. J., K. Klappe, I. Hummel, and J. W. Kok. 2004. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279: 5734-5738]. Here, we show that aminophospholipids are relatively enriched in Lubrol-resistant membrane domains compared with Triton X-100-resistant membrane domains, whereas sphingolipids are relatively enriched in the latter. Moreover, Lubrol-resistant membrane domains contain more protein and lipid mass. Based on these results, we postulate a model for detergent-insoluble glycosphingolipid-enriched membrane domains consisting of a Lubrol-insoluble/Triton X-100-insoluble region and a Lubrol-insoluble/Triton X-100-soluble region. The latter region contains most of the ABC transporters as well as lipids known to be necessary for their efflux activity. Compared with drug-sensitive cells, the detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in drug-resistant cells differ specifically in sphingolipid content and not in protein, phospholipid, or cholesterol content. In drug-resistant cells, sphingolipids with specific fatty acids (especially C24:1) are enriched in these membrane domains. Together, these data show that multidrug resistance-associated changes in both sphingolipids and ABC transporters occur in DIGs, but in different regions of these domains.

Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes.

PubMed

Lewis, Daniel R; Miller, Nathan D; Splitt, Bessie L; Wu, Guosheng; Spalding, Edgar P

2007-06-01

Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90 degrees reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.

AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.

PubMed

Zhang, Hui; Wang, Yi-Jun; Zhang, Yun-Kai; Wang, De-Shen; Kathawala, Rishil J; Patel, Atish; Talele, Tanaji T; Chen, Zhe-Sheng; Fu, Li-Wu

2014-08-01

AST1306, an inhibitor of EGFR and ErbB2, is currently in phase I of clinical trials. We evaluated the effect of AST306 on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters. We found that AST1306 significantly sensitized the ABC subfamily G member 2 (ABCG2)-overexpressing cells to ABCG2 substrate chemotherapeutics. AST1306 significantly increased intracellular accumulation of [(3)H]-mitoxantrone in ABCG2-overexpressing cells by blocking ABCG2 efflux function. Moreover, AST1306 stimulated the ATPase activity of ABCG2. Homology modeling predicted the binding conformation of AST1306 to be within the transmembrane region of ABCG2. In conclusion, AST1306 could notably reverse ABCG2-mediated MDR. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions.

PubMed

Szabó, Edit; Türk, Dóra; Telbisz, Ágnes; Kucsma, Nóra; Horváth, Tamás; Szakács, Gergely; Homolya, László; Sarkadi, Balázs; Várady, György

2018-01-01

ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. In this study we have examined the potential interactions of three key human ABC multidrug transporters with PhenGreen diacetate (PGD), a cell permeable fluorescent metal ion indicator. The non-fluorescent, hydrophobic PGD rapidly enters the cells and, after cleavage by cellular esterases, in the absence of quenching metal ions, PhenGreen (PG) becomes highly fluorescent. We found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. This fluorescence signal in the presence of specific transporter inhibitors is increased to the fluorescence levels in the control cells. Thus the PG accumulation assay is a new, unique tool for the parallel determination of the function of the ABCG2, ABCB1, and ABCC1 multidrug transporters. Since PG has very low cellular toxicity, the PG accumulation assay also allows the selection, separation and culturing of selected cell populations expressing either of these transporters.

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.

Catalytic and transport cycles of ABC exporters.

PubMed

Al-Shawi, Marwan K

2011-09-07

ABC (ATP-binding cassette) transporters are arguably the most important family of ATP-driven transporters in biology. Despite considerable effort and advances in determining the structures and physiology of these transporters, their fundamental molecular mechanisms remain elusive and highly controversial. How does ATP hydrolysis by ABC transporters drive their transport function? Part of the problem in answering this question appears to be a perceived need to formulate a universal mechanism. Although it has been generally hoped and assumed that the whole superfamily of ABC transporters would exhibit similar conserved mechanisms, this is proving not to be the case. Structural considerations alone suggest that there are three overall types of coupling mechanisms related to ABC exporters, small ABC importers and large ABC importers. Biochemical and biophysical characterization leads us to the conclusion that, even within these three classes, the catalytic and transport mechanisms are not fully conserved, but continue to evolve. ABC transporters also exhibit unusual characteristics not observed in other primary transporters, such as uncoupled basal ATPase activity, that severely complicate mechanistic studies by established methods. In this chapter, I review these issues as related to ABC exporters in particular. A consensus view has emerged that ABC exporters follow alternating-access switch transport mechanisms. However, some biochemical data suggest that alternating catalytic site transport mechanisms are more appropriate for fully symmetrical ABC exporters. Heterodimeric and asymmetrical ABC exporters appear to conform to simple alternating-access-type mechanisms.

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

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

ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mingli; Yin, Huancai; Bai, Pengli

This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity ofmore » QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.« less

Overcoming Multidrug Resistance in Human Cancer Cells by Natural Compounds

PubMed Central

Nabekura, Tomohiro

2010-01-01

Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural compounds, including (-)-epigallocatechin gallate, curcumin, capsaicin, and guggulsterone, as promising candidates. In this review, studies on the effects of natural compounds on P-glycoprotein and anticancer drug efflux transporters are summarized. PMID:22069634

Multidrug Resistance Proteins (MRPs/ABCCs) in Cancer Chemotherapy and Genetic Diseases

PubMed Central

Chen, Zhe-Sheng; Tiwari, Amit K.

2011-01-01

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the ‘C’ subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called Multidrug Resistance Proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C4 and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this review article, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed. PMID:21740521

Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide.

PubMed

Tournier, Nicolas; Saba, Wadad; Cisternino, Salvatore; Peyronneau, Marie-Anne; Damont, Annelaure; Goutal, Sébastien; Dubois, Albertine; Dollé, Frédéric; Scherrmann, Jean-Michel; Valette, Héric; Kuhnast, Bertrand; Bottlaender, Michel

2013-10-01

Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [(11)C]GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [(11)C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [(11)C]GLB uptake and its subsequent impact on [(11)C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [(11)C]GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet–Drug Interactions

PubMed Central

Telang, Urvi; Ji, Yan; Morris, Marilyn E.

2013-01-01

Isothiocyanates, a class of anti-cancer agents, are derived from cruciferous vegetables such as broccoli, cabbage and watercress, and have demonstrated chemopreventive activity in a number of cancer models and epidemiologic studies. Due to public interest in cancer prevention and alternative therapies in cancer, the consumption of herbal supplements and vegetables containing these compounds is widespread and increasing. Isothiocyanates interact with ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, MRP1, MRP2 and BCRP, and may influence the pharmacokinetics of substrates of these transporters. This review discusses the pharmacokinetic properties of isothiocyanates, their interactions with ABC transporters, and presents some data describing the potential for isothiocyanate-mediated diet–drug interactions. PMID:19623673

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

ABC-B transporter genes in Dirofilaria immitis.

PubMed

Bourguinat, Catherine; Che, Hua; Mani, Thangadurai; Keller, Kathy; Prichard, Roger K

2016-08-01

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite. Copyright © 2016. Published by Elsevier Ltd.

Multidrug efflux transporter, AcrB--the pumping mechanism.

PubMed

Murakami, Satoshi

2008-08-01

Resistance nodulation cell division (RND) transporters are one of the main causes of the bacterial multidrug resistance. They pump a wide range of antibiotics out of the cell by proton motive force. AcrB is the major RND transporter in Escherichia coli. Recently, the crystal structures of AcrB have been determined by different space groups. All these structures are consistent with asymmetric trimer. Each monomer has different conformation corresponding to one of the three functional states of the transport cycle. Transporting hydrophobic drug was bound in the periplasmic domain on one of the three monomers. The transport pathway with alternating access mechanism is located at the hydrophilic domain protruded into the periplasmic space while this mechanism of other transporter families like ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporter is located in the membrane-embedded region. For the RND, protonation might also take place asymmetrically at the functionally important charged residues in the transmembrane (TM) region. The structures indicate that drugs are transported by a three-step functional rotation in which substrates undergo ordered binding change.

ATP-induced conformational changes of nucleotide-binding domains in an ABC transporter. Importance of the water-mediated entropic force.

PubMed

Hayashi, Tomohiko; Chiba, Shuntaro; Kaneta, Yusuke; Furuta, Tadaomi; Sakurai, Minoru

2014-11-06

ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD-NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters.

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

PubMed Central

Greene, Nicholas P.; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

2018-01-01

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking. PMID:29892271

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

PubMed

Greene, Nicholas P; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

2018-01-01

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

2016-03-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

Effect of β-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

PubMed

Tang, Chao-Yuan; Zhu, Li-Xin; Yu, Jian-Dong; Chen, Zhi; Gu, Man-Cang; Mu, Chao-Feng; Liu, Qi; Xiong, Yang

2018-07-30

In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by β-elemene (β-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by β-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOX Fluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOX Fluc cells being treated with β-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOX Fluc was lessened when pretreated with β-ELE, which means that β-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of β-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of β-ELE. To verify the efficacy of β-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and β-ELE. MTT assay showed that β-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC 50 of the combination group was much lower than that of the single DOX or β-ELE treatment. In all, β-ELE may reverse MDR through the substrates of ABC transporters

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

USDA-ARS?s Scientific Manuscript database

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...

Placental ABC Transporters: Biological Impact and Pharmaceutical Significance.

PubMed

Joshi, Anand A; Vaidya, Soniya S; St-Pierre, Marie V; Mikheev, Andrei M; Desino, Kelly E; Nyandege, Abner N; Audus, Kenneth L; Unadkat, Jashvant D; Gerk, Phillip M

2016-12-01

The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

Placental ABC Transporters: Biological Impact and Pharmaceutical Significance

PubMed Central

Joshi, Anand A.; Vaidya, Soniya S.; St-Pierre, Marie V.; Mikheev, Andrei M.; Desino, Kelly E.; Nyandege, Abner N.; Audus, Kenneth L.; Unadkat, Jashvant D.; Gerk, Phillip M.

2017-01-01

The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy. PMID:27644937

The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo.

PubMed

Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

2012-07-01

The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me-/- mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me-/- erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me-/- erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me-/- erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo.

The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo

PubMed Central

Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

2012-01-01

The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me−/− mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me−/− erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me−/− erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me−/− erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo. PMID:22240895

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

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

Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance

PubMed Central

Wang, Yi-Jun; Zhang, Yun-Kai; Kathawala, Rishil J.; Chen, Zhe-Sheng

2014-01-01

The phenomenon of multidrug resistance (MDR) has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC) transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs), such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance. PMID:25268163

EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry.

PubMed

Rotem, Dvir; Schuldiner, Shimon

2004-11-19

Multidrug transporters recognize and transport substrates with apparently little common structural features. At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges. Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells. Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins. Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein. The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e. electrogenic), the transport of divalent substrate does not (i.e. electroneutral). Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle. In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones. In the presence of proton gradients, resistance to both types of substrates is detected. The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance.

Evolutionary Trajectories of Entomopathogenic Fungi ABC Transporters.

PubMed

Baral, Bikash

2017-01-01

The ABC protein superfamily-also called traffic ATPases-are energy-dependent ubiquitous proteins, representing one of the crucial and the largest family in the fungal genomes. The ATP-binding cassette endows a characteristic 200-250 amino acids and is omnipresent in all organisms ranging from prokaryotes to eukaryotes. Unlike in bacteria with nutrient import functions, ABC transporters in fungal entomopathogens serve as effective efflux pumps that are largely involved in the shuttle of metabolites across the biological membranes. Thus, the search for ABC proteins may prove of immense importance in elucidating the functional and molecular mechanism at the host-pathogen (insect-fungus) interface. Their sequence homology, domain topology, and functional traits led to the actual identification of nine different families in fungal entomopathogens. Evolutionary relationships within the ABC superfamily are discussed, concentrating on computational approaches for comparative identification of ABC transporters in insect-pathogenic fungi (entomopathogens) with those of animals, plants, and their bacterial orthologs. Ancestors of some fungal candidates have duplicated extensively in some phyla, while others were lost in one lineage or the other, and predictions for the cause of their duplications and/or loss in some phyla are made. ABC transporters of fungal insect-pathogens serve both defensive and offensive functions effective against land-dwelling and ground foraging voracious insects. This study may help to unravel the molecular cascades of ABC proteins to illuminate the means through which insects cope with fungal infection and fungal-related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple molecular mechanisms for multidrug resistance transporters.

PubMed

Higgins, Christopher F

2007-04-12

The acquisition of multidrug resistance is a serious impediment to improved healthcare. Multidrug resistance is most frequently due to active transporters that pump a broad spectrum of chemically distinct, cytotoxic molecules out of cells, including antibiotics, antimalarials, herbicides and cancer chemotherapeutics in humans. The paradigm multidrug transporter, mammalian P-glycoprotein, was identified 30 years ago. Nonetheless, success in overcoming or circumventing multidrug resistance in a clinical setting has been modest. Recent structural and biochemical data for several multidrug transporters now provide mechanistic insights into how they work. Organisms have evolved several elegant solutions to ridding the cell of such cytotoxic compounds. Answers are emerging to questions such as how multispecificity for different drugs is achieved, why multidrug resistance arises so readily, and what chance there is of devising a clinical solution.

The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

PubMed

Martin, Audrey; Daniel, Jaiyanth

2018-02-05

Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function. Copyright © 2018 Elsevier Inc. All rights reserved.

Montelukast is a potent and durable inhibitor of multidrug resistance protein 2 (MRP2)-mediated efflux of taxol and saquinavir

PubMed Central

Roy, Upal; Chakravarty, Geetika; Honer Zu Bentrup, Kerstin; Mondal, Debasis

2009-01-01

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of HIV-1 protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-gp and MRP2, are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair™) and zafirlukast (Accolate™), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 μM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [3H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 mins post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells. PMID:19952419

ABC transporters and immunity: mechanism of self-defense.

PubMed

Hinz, Andreas; Tampé, Robert

2012-06-26

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.

Diversity in ABC transporters: Type I, II and III importers

PubMed Central

Rice, Austin J.; Park, Aekyung

2014-01-01

ATP-binding cassette transporters are multi-subunit membrane pumps that transport substrates across membranes. While significant in the transport process, transporter architecture exhibits a range of diversity that we are only beginning to recognize. This divergence may provide insight into the mechanisms of substrate transport and homeostasis. Until recently, ABC importers have been classified into two types, but with the emergence of energy-coupling factor (ECF) transporters there are potentially three types of ABC importers. In this review, we summarize an expansive body of research on the three types of importers with an emphasis on the basics that underlie ABC importers, such as structure, subunit composition and mechanism. PMID:25155087

Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCG2.

PubMed

Murakami, Megumi; Ohnuma, Shinobu; Fukuda, Michihiro; Chufan, Eduardo E; Kudoh, Katsuyoshi; Kanehara, Keigo; Sugisawa, Norihiko; Ishida, Masaharu; Naitoh, Takeshi; Shibata, Hiroyuki; Iwabuchi, Yoshiharu; Ambudkar, Suresh V; Unno, Michiaki

2017-11-01

Multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) transporters in cancer cells is a major obstacle in cancer chemotherapy. Previous studies have shown that curcumin, a natural product and a dietary constituent of turmeric, inhibits the function of MDR-related ABC transporters, including ABCB1, ABCC1, and especially ABCG2. However, the limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinical setting. In this study, we investigated the effects of 24 synthetic curcumin analogs with increased bioavailability on the transport function of ABCG2. The screening of the 24 synthetic analogs by means of flow cytometry revealed that four of the curcumin analogs (GO-Y030, GO-Y078, GO-Y168, and GO-Y172) significantly inhibited the efflux of the ABCG2 substrates, mitoxantrone and pheophorbide A, from ABCG2-overexpressing K562/breast cancer resistance protein (BCRP) cells. Biochemical analyses showed that GO-Y030, GO-Y078, and GO-Y172 stimulated the ATPase activity of ABCG2 at nanomolar concentrations and inhibited the photolabeling of ABCG2 with iodoarylazidoprazosin, suggesting that these analogs interact with the substrate-binding sites of ABCG2. In addition, when used in cytotoxicity assays, GO-Y030 and GO-Y078 were found to improve the sensitivity of the anticancer drug, SN-38, in K562/BCRP cells. Taken together, these results suggest that nontoxic synthetic curcumin analogs with increased bioavailability, especially GO-Y030 and GO-Y078, inhibit the function of ABCG2 by directly interacting at the substrate-binding site. These synthetic curcumin analogs could therefore be developed as potent modulators to overcome ABCG2-mediated MDR in cancer cells. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika

2011-01-07

Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplishedmore » by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte

Multidrug Resistance in Breast Cancer: From In Vitro Models to Clinical Studies

PubMed Central

Wind, N. S.; Holen, I.

2011-01-01

The development of multidrug resistance (MDR) and subsequent relapse on therapy is a widespread problem in breast cancer, but our understanding of the underlying molecular mechanisms is incomplete. Numerous studies have aimed to establish the role of drug transporter pumps in MDR and to link their expression to response to chemotherapy. The ATP-binding cassette (ABC) transporters are central to breast cancer MDR, and increases in ABC expression levels have been shown to correlate with decreases in response to various chemotherapy drugs and a reduction in overall survival. But as there is a large degree of redundancy between different ABC transporters, this correlation has not been seen in all studies. This paper provides an introduction to the key molecules associated with breast cancer MDR and summarises evidence of their potential roles reported from model systems and clinical studies. We provide possible explanations for why despite several decades of research, the precise role of ABC transporters in breast cancer MDR remains elusive. PMID:22332018

ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

PubMed

Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

2018-03-08

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM

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.

A multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis japonica.

PubMed

Takanashi, Kojiro; Yamada, Yasuyuki; Sasaki, Takayuki; Yamamoto, Yoko; Sato, Fumihiko; Yazaki, Kazufumi

2017-06-01

Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications.

PubMed

Stockner, Thomas; Mullen, Anna; MacMillan, Fraser

2015-10-01

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented. © 2015 Authors; published by Portland Press Limited.

Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function.

PubMed

Feng, Sen-Ling; Yuan, Zhong-Wen; Yao, Xiao-Jun; Ma, Wen-Zhe; Liu, Liang; Liu, Zhong-Qiu; Xie, Ying

2016-08-01

Multidrug resistance (MDR) and tumor metastasis are the main causes of chemotherapeutic treatment failure and mortality in cancer patients. In this study, at achievable nontoxic plasma concentrations, citrus flavonoid tangeretin has been shown to reverse ABCB1-mediated cancer resistance to a variety of chemotherapeutic agents effectively. Co-treatment of cells with tangeretin and paclitaxel activated apoptosis as well as arrested cell cycle at G2/M-phase. Tangeretin profoundly inhibited the ABCB1 transporter activity since it significantly increased the intracellular accumulation of doxorubicin, and flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the expression of ABCB1. Moreover, it stimulated the ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. The molecular docking results indicated a favorable binding of tangeretin with the transmemberane region site 1 of homology modeled ABCB1 transporter. The overall results demonstrated that tangeretin could sensitize ABCB1-overexpressing cancer cells to chemotherapeutical agents by directly inhibiting ABCB1 transporter function, which encouraged further animal and clinical studies in the treatment of resistant cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

PubMed

Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

2016-03-22

Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

PubMed Central

Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

2010-01-01

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

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

PubMed

Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

2017-09-20

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

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

PubMed Central

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

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

ABC transporters are involved in defense against permethrin insecticide in the malaria vector Anopheles stephensi.

PubMed

Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Comandatore, Francesco; Sassera, Davide; Rossi, Paolo; Cafarchia, Claudia; Otranto, Domenico; Favia, Guido; Genchi, Claudio; Bandi, Claudio; Urbanelli, Sandra

2014-07-29

Proteins from the ABC family (ATP-binding cassette) represent the largest known group of efflux pumps, responsible for transporting specific molecules across lipid membranes in both prokaryotic and eukaryotic organisms. In arthropods they have been shown to play a role in insecticide defense/resistance. The presence of ABC transporters and their possible association with insecticide transport have not yet been investigated in the mosquito Anopheles stephensi, the major vector of human malaria in the Middle East and South Asian regions. Here we investigated the presence and role of ABCs in transport of permethrin insecticide in a susceptible strain of this mosquito species. To identify ABC transporter genes we obtained a transcriptome from untreated larvae of An. stephensi and then compared it with the annotated transcriptome of Anopheles gambiae. To analyse the association between ABC transporters and permethrin we conducted bioassays with permethrin alone and in combination with an ABC inhibitor, and then we investigated expression profiles of the identified genes in larvae exposed to permethrin. Bioassays showed an increased mortality of mosquitoes when permethrin was used in combination with the ABC-transporter inhibitor. Genes for ABC transporters were detected in the transcriptome, and five were selected (AnstABCB2, AnstABCB3, AnstABCB4, AnstABCmember6 and AnstABCG4). An increased expression in one of them (AnstABCG4) was observed in larvae exposed to the LD50 dose of permethrin. Contrary to what was found in other insect species, no up-regulation was observed in the AnstABCB genes. Our results show for the first time the involvement of ABC transporters in larval defense against permethrin in An. stephensi and, more in general, confirm the role of ABC transporters in insecticide defense. The differences observed with previous studies highlight the need of further research as, despite the growing number of studies on ABC transporters in insects, the

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

PubMed Central

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

2013-01-01

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

Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Nieves-Morión, Mercedes; Flores, Enrique

2018-02-01

Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Structural basis for the blockade of MATE multidrug efflux pumps

DOE PAGES

Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; ...

2015-08-06

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H + or Na + electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H +-coupled DinF transporter, as well as of an H +-coupled DinF and a Na +-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm themore » biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less

Diversity of ABC transporter genes across the plant kingdom and their potential utility in biotechnology.

PubMed

Lane, Thomas S; Rempe, Caroline S; Davitt, Jack; Staton, Margaret E; Peng, Yanhui; Soltis, Douglas Edward; Melkonian, Michael; Deyholos, Michael; Leebens-Mack, James H; Chase, Mark; Rothfels, Carl J; Stevenson, Dennis; Graham, Sean W; Yu, Jun; Liu, Tao; Pires, J Chris; Edger, Patrick P; Zhang, Yong; Xie, Yinlong; Zhu, Ying; Carpenter, Eric; Wong, Gane Ka-Shu; Stewart, C Neal

2016-05-31

The ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity. We utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p < 0.005), and green algae, red algae, and bryophytes had significantly more ABCF transporter gene members (p < 0.005). Ferns had significantly fewer ABCA transporter gene members than all other plant groups (p < 0.005). We present a transcriptomic overview of ABC transporter gene members across all major plant groups. An increase in the number of gene family members present in the ABCB, ABCC, and ABCD transporter subfamilies may indicate an expansion of the ABC transporter superfamily among green land plants, which include all crop species. The striking difference between the number of ABCA subfamily transporter

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

PubMed

Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

2015-02-10

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. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump

PubMed Central

Llabrés, Salomé; Neuberger, Arthur; Blaza, James N.; Bai, Xiao-chen; Okada, Ui; Murakami, Satoshi; van Veen, Hendrik W.; Zachariae, Ulrich; Scheres, Sjors H.W.; Luisi, Ben F.

2017-01-01

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain (TMD) with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism. PMID:28504659

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

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning

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 ABCmore » 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.« less

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

PubMed

van der Heide, T; Poolman, B

2000-06-20

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane

PubMed Central

van der Heide, Tiemen; Poolman, Bert

2000-01-01

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein–lipid interactions. PMID:10860977

Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plants

PubMed Central

Remy, Estelle; Duque, Paula

2014-01-01

Higher plants possess a multitude of Multiple Drug Resistance (MDR) transporter homologs that group into three distinct and ubiquitous families—the ATP-Binding Cassette (ABC) superfamily, the Major Facilitator Superfamily (MFS), and the Multidrug And Toxic compound Extrusion (MATE) family. As in other organisms, such as fungi, mammals, and bacteria, MDR transporters make a primary contribution to cellular detoxification processes in plants, mainly through the extrusion of toxic compounds from the cell or their sequestration in the central vacuole. This review aims at summarizing the currently available information on the in vivo roles of MDR transporters in plant systems. Taken together, these data clearly indicate that the biological functions of ABC, MFS, and MATE carriers are not restricted to xenobiotic and metal detoxification. Importantly, the activity of plant MDR transporters also mediates biotic stress resistance and is instrumental in numerous physiological processes essential for optimal plant growth and development, including the regulation of ion homeostasis and polar transport of the phytohormone auxin. PMID:24910617

ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma

PubMed Central

2013-01-01

Background Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. Methods Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. Results Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. Conclusions ABC transporter inhibitors are already being

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

PubMed Central

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

2013-01-01

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. PMID:23341609

The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin

PubMed Central

2004-01-01

Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [3H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin–Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [3H]UCB uptake was measured in the presence of 60 μM human serum albumin at a free (unbound) concentration of UCB (BF) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5′-[β,γ-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [3H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 μM MK571 and followed saturative kinetics with Km=10±3 nM (BF) and Vmax=100±13 pmol·min−1·(mg of protein)−1. UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity. PMID:15245331

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

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

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

PubMed

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

2016-01-01

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

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Alectinib (CH5424802) antagonizes ABCB1- and ABCG2-mediated multidrug resistance in vitro, in vivo and ex vivo.

PubMed

Yang, Ke; Chen, Yifan; To, Kenneth Kin Wah; Wang, Fang; Li, Delan; Chen, Likun; Fu, Liwu

2017-03-17

Alectinib, an inhibitor of anaplastic lymphoma kinase (ALK), was approved by the Food and Drug Administration (FDA) for the treatment of patients with ALK-positive non-small cell lung cancer (NSCLC). Here we investigated the reversal effect of alectinib on multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters, which is the primary cause of chemotherapy failure. We provide the first evidence that alectinib increases the sensitivity of ABCB1- and ABCG2-overexpressing cells to chemotherapeutic agents in vitro and in vivo. Mechanistically, alectinib increased the intracellular accumulation of ABCB1/ABCG2 substrates such as doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, alectinib stimulated ATPase activity and competed with substrates of ABCB1 or ABCG2 and competed with [125I] iodoarylazidoprazosin (IAAP) photolabeling bound to ABCB1 or ABCG2 but neither altered the expression and localization of ABCB1 or ABCG2 nor the phosphorylation levels of AKT and ERK. Alectinib also enhanced the cytotoxicity of DOX and the intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. These findings suggest that alectinib combined with traditional chemotherapy may be beneficial to patients with ABCB1- or ABCG2-mediated MDR.

Alectinib (CH5424802) antagonizes ABCB1- and ABCG2-mediated multidrug resistance in vitro, in vivo and ex vivo

PubMed Central

Yang, Ke; Chen, Yifan; To, Kenneth Kin Wah; Wang, Fang; Li, Delan; Chen, Likun; Fu, Liwu

2017-01-01

Alectinib, an inhibitor of anaplastic lymphoma kinase (ALK), was approved by the Food and Drug Administration (FDA) for the treatment of patients with ALK-positive non-small cell lung cancer (NSCLC). Here we investigated the reversal effect of alectinib on multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters, which is the primary cause of chemotherapy failure. We provide the first evidence that alectinib increases the sensitivity of ABCB1- and ABCG2-overexpressing cells to chemotherapeutic agents in vitro and in vivo. Mechanistically, alectinib increased the intracellular accumulation of ABCB1/ABCG2 substrates such as doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, alectinib stimulated ATPase activity and competed with substrates of ABCB1 or ABCG2 and competed with [125I] iodoarylazidoprazosin (IAAP) photolabeling bound to ABCB1 or ABCG2 but neither altered the expression and localization of ABCB1 or ABCG2 nor the phosphorylation levels of AKT and ERK. Alectinib also enhanced the cytotoxicity of DOX and the intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. These findings suggest that alectinib combined with traditional chemotherapy may be beneficial to patients with ABCB1- or ABCG2-mediated MDR. PMID:28303028

The enriched fraction of Vernonia cinerea L. induces apoptosis and inhibits multi-drug resistance transporters in human epithelial cancer cells.

PubMed

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

2014-12-02

Vernonia cinerea Less. (VC) of the family Asteraceaes is considered as the sacred plant; 'Dasapushpam' which is ethnopharmacologically significant to the people of Kerala in India. In fact, VC has been used in the traditional system of medicine (Ayurveda) for the treatment of various ailments including cancer. Cytotoxicity of the ethanolic extract of VC (VC-ET), petroleum ether fraction (VC-PET), dichloromethane fraction (VC-DCM), n-butyl alcohol fraction (VC-BT), and rest fraction (VC-R) was evaluated in cervical carcinoma (HeLa), lung adenocarcinoma (A549), breast cancer (MCF-7), and colon carcinoma (Caco-2) cells using Sulforhodamine B (SRB) assay. The apoptotic effects of VC-DCM were assessed in cancer cells using Annexin V assay. The effects of VC-DCM on multi-drug resistance (MDR) transporters in HeLa, A549, MCF-7, and Caco-2 cells were evaluated using flow cytometry based functional assays. Similarly, drug uptake in cancer cells and sensitization of cancer cells towards chemotherapeutic drugs in the presence of VC-DCM were studied using Daunorubicin (DNR) accumulation assay and SRB assay, respectively. Cytotoxicity assay revealed that the enriched fraction of VC (VC-DCM) possessed dose-dependent cytotoxic effects in human epithelial cancer cells (HeLa, A549, MCF-7, and Caco-2). Further, treatment of cancer cells (HeLa, A549, MCF-7, and Caco-2) with VC-DCM led to a significant increase in both early and late apoptosis, indicating the induction of apoptosis. Interestingly, VC-DCM significantly inhibited functional activity of MDR transporters (ABC-B1 and ABC-G2), enhanced DNR-uptake in cancer cells, and sensitized cancer cells towards chemotherapeutic drug-mediated cytotoxicity, thus indicating the ability of VC-DCM to reverse MDR in cancer and enhance the cytotoxic effects of anticancer drugs. A methodological investigation on the anti-cancer properties of Vernonia cinerea Less. (VC) revealed that an enriched fraction of VC (VC-DCM) possessed cytotoxic

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death*

PubMed Central

Chowdhury, Sayan; Mukhopadhyay, Rupkatha; Saha, Sourav; Mishra, Amartya; Sengupta, Souvik; Roy, Syamal; Majumder, Hemanta K.

2014-01-01

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB25R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance. PMID:24706751

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro

PubMed Central

Kievit, Forrest M.; Wang, Freddy Y.; Fang, Chen; Mok, Hyejung; Wang, Kui; Silber, John R.; Ellenbogen, Richard G.; Zhang, Miqin

2011-01-01

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR. PMID:21277920

Multidrug and toxin extrusion proteins as transporters of antimicrobial drugs.

PubMed

Nies, Anne T; Damme, Katja; Schaeffeler, Elke; Schwab, Matthias

2012-12-01

Antimicrobial drugs are essential in the treatment of infectious diseases. A better understanding of transport processes involved in drug disposition will improve the predictability of drug-drug interactions with consequences for drug response. Multidrug And Toxin Extrusion (MATE; SLC47A) proteins are efflux transporters mediating the excretion of several antimicrobial drugs as well as other organic compounds into bile and urine, thereby contributing to drug disposition. This review summarizes current knowledge of the structural and molecular features of human MATE transporters including their functional role in drug transport with a specific focus on antimicrobial drugs. The PubMed database was searched using the terms "MATE1," "MATE-2K," "MATE2," "SLC47A1," "SLC47A2," and "toxin extrusion protein" (up to June 2012). MATE proteins have been recognized as important transporters mediating the final excretion step of cationic drugs into bile and urine. These include the antiviral drugs acyclovir, amprenavir, and ganciclovir, the antibiotics cephalexin, cephradine and levofloxacin, as well as the antimalarial agents chloroquine and quinine. It is therefore important to enhance our understanding of the role of MATEs in drug extrusion with particular emphasis on the functional consequences of genetic variants on disposition of these antimicrobial drugs.

ABCdb: an online resource for ABC transporter repertories from sequenced archaeal and bacterial genomes.

PubMed

Fichant, Gwennaele; Basse, Marie-Jeanne; Quentin, Yves

2006-03-01

The ATP-binding cassette (ABC) transporters are one of the major classes of active transporters. They are widespread in archaea, bacteria, and eukaryota, indicating that they have arisen early in evolution. They are involved in many essential physiological processes, but the majority import or export a wide variety of compounds across cellular membranes. These systems share a common architecture composed of four (exporters) or five (importers) domains. To identify and reconstruct functional ABC transporters encoded by archaeal and bacterial genomes, we have developed a bioinformatic strategy. Cross-reference to the transport classification system is used to predict the type of compound transported. A high quality of annotation is achieved by manual verification of the predictions. However, in order to face the rapid increase in the number of published genomes, we also include analyses of genomes issuing directly from the automated strategy. Querying the database (http://www-abcdb.biotoul.fr) allows to easily retrieve ABC transporter repertories and related data. Additional query tools have been developed for the analysis of the ABC family from both functional and evolutionary perspectives.

Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

PubMed

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

2015-11-03

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

Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

PubMed

Liu, Su; Jiang, Wei; Wu, Bing; Yu, Jing; Yu, Haiyan; Zhang, Xu-Xiang; Torres-Duarte, Cristina; Cherr, Gary N

2016-01-01

Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 μg/mL) and GO (<20 μg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

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. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking.

PubMed

Dai, Fuhong; Yoo, Won Gi; Lee, Ji-Yun; Lu, Yanyan; Pak, Jhang Ho; Sohn, Woon-Mok; Hong, Sung-Jong

2017-11-21

Multidrug resistance-associated protein 4 (MRP4) is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC) transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host's bile duct, accumulation of bile juice can be toxic to the worm's tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body. We cloned MRP4 (CsMRP4) from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2) and two nucleotide-binding domains (NBD1 & 2) as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa) fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults. Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites' survival.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

PubMed

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

2011-01-07

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded. Copyright © 2010 Elsevier Inc. All rights reserved.

Rational design of cancer-targeted selenium nanoparticles to antagonize multidrug resistance in cancer cells.

PubMed

Liu, Ting; Zeng, Lilan; Jiang, Wenting; Fu, Yuanting; Zheng, Wenjie; Chen, Tianfeng

2015-05-01

Multidrug resistance is one of the greatest challenges in cancer therapy. Herein we described the synthesis of folate (FA)-conjugated selenium nanoparticles (SeNPs) as cancer-targeted nano-drug delivery system for ruthenium polypyridyl (RuPOP) exhibits strong fluorescence, which allows the direct imaging of the cellular trafficking of the nanosystem. This nanosystem could effectively antagonize against multidrug resistance in liver cancer. FA surface conjugation significantly enhanced the cellular uptake of SeNPs by FA receptor-mediated endocytosis through nystain-dependent lipid raft-mediated and clathrin-mediated pathways. The nanomaterials overcame the multidrug resistance in R-HepG2 cells through inhibition of ABC family proteins expression. Internalized nanoparticles triggered ROS overproduction and induced apoptosis by activating p53 and MAPKs pathways. Moreover, FA-SeNPs exhibited low in vivo acute toxicity, which verified the safety and application potential of FA-SeNPs as nanodrugs. This study provides an effective strategy for the design of cancer-targeted nanodrugs against multidrug resistant cancers. In the combat against hepatocellular carcinoma, multidrug resistance remains one of the obstacles to be overcome. The authors designed and synthesized folate (FA)-conjugated selenium nanoparticles (SeNPs) with enhanced cancer-targeting capability. This system carried ruthenium polypyridyl (RuPOP), an efficient metal-based anti-cancer drug with strong fluorescence. It was shown that this combination was effective in antagonizing against multidrug resistance in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

PubMed

Guerreiro, Denise Damasceno; de Lima, Laritza Ferreira; Mbemya, Gildas Tetaping; Maside, Carolina Mielgo; Miranda, André Marrocos; Tavares, Kaio César Simiano; Alves, Benner Geraldo; Faustino, Luciana Rocha; Smitz, Johan; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2018-06-01

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

ATP-binding cassette (ABC) proteins in aquatic invertebrates: Evolutionary significance and application in marine ecotoxicology.

PubMed

Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Jae-Seong

2017-04-01

The ATP-binding cassette (ABC) protein superfamily is known to play a fundamental role in biological processes and is highly conserved across animal taxa. The ABC proteins function as active transporters for multiple substrates across the cellular membrane by ATP hydrolysis. As this superfamily is derived from a common ancestor, ABC genes have evolved via lineage-specific duplications through the process of adaptation. In this review, we summarized information about the ABC gene families in aquatic invertebrates, considering their evolution and putative functions in defense mechanisms. Phylogenetic analysis was conducted to examine the evolutionary significance of ABC gene families in aquatic invertebrates. Particularly, a massive expansion of multixenobiotic resistance (MXR)-mediated efflux transporters was identified in the absence of the ABCG2 (BCRP) gene in Ecdysozoa and Platyzoa, suggesting that a loss of Abcg2 gene occurred sporadically in these species during divergence of Protostome to Lophotrochozoa. Furthermore, in aquatic invertebrates, the ecotoxicological significance of MXR is discussed while considering the role of MXR-mediated efflux transporters in response to various environmental pollutants. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Induction of hepatic ABC transporter expression is part of the PPARalpha-mediated fasting response in the mouse.

PubMed

Kok, Tineke; Wolters, Henk; Bloks, Vincent W; Havinga, Rick; Jansen, Peter L M; Staels, Bart; Kuipers, Folkert

2003-01-01

Fatty acids are natural ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPARalpha-dependent manner. A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Pparalpha((-/-)) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components. Expression of Pparalpha, farnesoid X receptor, and liver X receptor alpha genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Pparalpha((-/-)) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%. Induction of Mdr2 expression and function is part of the PPARalpha-mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis.

PubMed

Qiu, Jingfan; Zhuo, Ying; Zhu, Dongqing; Zhou, Xiufen; Zhang, Lixin; Bai, Linquan; Deng, Zixin

2011-10-01

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

Interaction of Food Additives with Intestinal Efflux Transporters.

PubMed

Sjöstedt, Noora; Deng, Feng; Rauvala, Oskari; Tepponen, Tuomas; Kidron, Heidi

2017-11-06

Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2) and P-glycoprotein (P-gp) are ABC transporters that are expressed in the intestine, where they are involved in the efflux of many drugs from enterocytes back into the intestinal lumen. The inhibition of BCRP, MRP2, and P-gp can result in enhanced absorption and exposure of substrate drugs. Food additives are widely used by the food industry to improve the stability, flavor, and consistency of food products. Although they are considered safe for consumption, their interactions with intestinal transporters are poorly characterized. Therefore, in this study, selected food additives, including preservatives, colorants, and sweeteners, were studied in vitro for their inhibitory effects on intestinal ABC transporters. Among the studied compounds, several colorants were able to inhibit BCRP and MRP2, whereas P-gp was fairly insensitive to inhibition. Additionally, one sweetener was identified as a potent inhibitor of BCRP. Dose-response studies revealed that the IC 50 values of the inhibitors were lower than the estimated intestinal concentrations after the consumption of beverages containing food colorants. This suggests that there is potential for previously unrecognized transporter-mediated food additive-drug interactions.

Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters

PubMed Central

Andreoletti, Pierre; Raas, Quentin; Gondcaille, Catherine; Cherkaoui-Malki, Mustapha; Trompier, Doriane; Savary, Stéphane

2017-01-01

The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85  Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues. PMID:28737695

Osimertinib (AZD9291), a Mutant-Selective EGFR Inhibitor, Reverses ABCB1-Mediated Drug Resistance in Cancer Cells.

PubMed

Zhang, Xiao-Yu; Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Zeng, Leli; Xu, Megan; Wang, Xiu-Qi; Yang, Dong-Hua; Chen, Zhe-Sheng

2016-09-15

In recent years, tyrosine kinase inhibitors (TKIs) have been shown capable of inhibiting the ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR). In this study, we determine whether osimertinib, a novel selective, irreversible EGFR (epidermal growth factor receptor) TKI, could reverse ABC transporter-mediated MDR. The results showed that, at non-toxic concentrations, osimertinib significantly sensitized both ABCB1-transfected and drug-selected cell lines to substrate anticancer drugs colchicine, paclitaxel, and vincristine. Osimertinib significantly increased the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant alteration in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 0.3 µM osimertinib for 72 h. In addition, ATPase assay showed osimertinib stimulated ABCB1 ATPase activity. Molecular docking and molecular dynamic simulations showed osimertinib has strong and stable interactions at the transmembrane domain of human homology ABCB1. Taken together, our findings suggest that osimertinib, a clinically-approved third-generation EGFR TKI, can reverse ABCB1-mediated MDR, which supports the combination therapy with osimertinib and ABCB1 substrates may potentially be a novel therapeutic stategy in ABCB1-positive drug resistant cancers.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

PubMed

Lüders, Ann-Katrin; Saborowski, Reinhard; Bickmeyer, Ulf

2009-09-01

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

PubMed

Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

2017-12-01

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

PubMed

Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

2016-04-01

For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux*

PubMed Central

Radchenko, Martha; Nie, Rongxin; Lu, Min

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism. PMID:26975373

Transporter-mediated natural product-drug interactions for the treatment of cardiovascular diseases.

PubMed

Zha, Weibin

2018-04-01

The growing use of natural products in cardiovascular (CV) patients has been greatly raising the concerns about potential natural product-CV drug interactions. Some of these may lead to unexpected cardiovascular adverse effects and it is, therefore, essential to identify or predict potential natural product-CV drug interactions, and to understand the underlying mechanisms. Drug transporters are important determinants for the pharmacokinetics of drugs and alterations of drug transport has been recognized as one of the major causes of natural product-drug interactions. In last two decades, many CV drugs (e.g., angiotensin II receptor blockers, beta-blockers and statins) have been identified to be substrates and inhibitors of the solute carrier (SLC) transporters and the ATP-binding cassette (ABC) transporters, which are two major transporter superfamilies. Meanwhile, in vitro and in vivo studies indicate that a growing number of natural products showed cardioprotective effects (e.g., gingko biloba, danshen and their active ingredients) are also substrates and inhibitors of drug transporters. Thus, to understand transporter-mediated natural product-CV drug interactions is important and some transporter-mediated interactions have already shown to have clinical relevance. In this review, we review the current knowledge on the role of ABC and SLC transporters in CV therapy, as well as transporter modulation by natural products used in CV diseases and their induced natural product-CV drug interactions through alterations of drug transport. We hope our review will aid in a comprehensive summary of transporter-mediated natural product-CV drug interactions and help public and physicians understand these type of interactions. Copyright © 2017. Published by Elsevier B.V.

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

Inhibition of the Human ABC Efflux Transporters P-gp and ...

EPA Pesticide Factsheets

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 [(3H)-paclitaxel and (3H)-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.

Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.

PubMed

Madoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J M; Thunnissen, Andy-Mark W H

2009-01-21

LmrR is a PadR-related transcriptional repressor that regulates the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topology containing a typical beta-winged helix-turn-helix domain with an additional C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer centre serving as a multidrug-binding site. The drugs bind in a similar manner with their aromatic rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.

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

Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi.

PubMed

Valenzuela-Muñoz, Valentina; Sturm, Armin; Gallardo-Escárate, Cristian

2015-04-09

ATP-binding cassette (ABC) protein family encode for membrane proteins involved in the transport of various biomolecules through the cellular membrane. These proteins have been identified in all taxa and present important physiological functions, including the process of insecticide detoxification in arthropods. For that reason the ectoparasite Caligus rogercresseyi represents a model species for understanding the molecular underpinnings involved in insecticide drug resistance. llumina sequencing was performed using sea lice exposed to 2 and 3 ppb of deltamethrin and azamethiphos. Contigs obtained from de novo assembly were annotated by Blastx. RNA-Seq analysis was performed and validated by qPCR analysis. From the transcriptome database of C. rogercresseyi, 57 putative members of ABC protein sequences were identified and phylogenetically classified into the eight subfamilies described for ABC transporters in arthropods. Transcriptomic profiles for ABC proteins subfamilies were evaluated throughout C. rogercresseyi development. Moreover, RNA-Seq analysis was performed for adult male and female salmon lice exposed to the delousing drugs azamethiphos and deltamethrin. High transcript levels of the ABCB and ABCC subfamilies were evidenced. Furthermore, SNPs mining was carried out for the ABC proteins sequences, revealing pivotal genomic information. The present study gives a comprehensive transcriptome analysis of ABC proteins from C. rogercresseyi, providing relevant information about transporter roles during ontogeny and in relation to delousing drug responses in salmon lice. This genomic information represents a valuable tool for pest management in the Chilean salmon aquaculture industry.

Kinetic control of TolC recruitment by multidrug efflux complexes.

PubMed

Tikhonova, Elena B; Dastidar, Vishakha; Rybenkov, Valentin V; Zgurskaya, Helen I

2009-09-22

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.

Insights on Na(+) binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM.

PubMed

Song, Jianing; Ji, Changge; Zhang, John Z H

2014-02-01

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+-substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild-type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na(+) ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di-Na(+) binding state is likely more efficient for conformational changes of NorM_VC toward the inward-facing conformation than single-Na(+) binding state. The observation of two Na(+) binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na(+) binding and enrich our knowledge of the transport mechanism of secondary transporters. Copyright © 2013 Wiley Periodicals, Inc.

Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force*

PubMed Central

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W.

2014-01-01

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na+ or H+. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na+ or H+ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. PMID:24711447

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.

Multidrug and toxin extrusion proteins mediate cellular transport of cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hong; Guo, Dong; Obianom, Obinna N.

Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd{sup 2+}). In this study, we aimed to examine whether Cd{sup 2+} is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd{sup 2+}. The cells overexpressing MATEs showed a 2–4more » fold increase of Cd{sup 2+} uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (K{sub m}) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd{sup 2+} could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC{sub 50}) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd{sup 2+} out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd{sup 2+}-induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd{sup 2+} and may function as cellular elimination machinery in Cd intoxication. - Highlights: • Cadmium is an environmentally prevalent toxicant. • Little was known regarding the elimination and detoxification of cadmium. • Cadmium ion is here demonstrated as a substrate of MATE transporters. • MATEs may function as cellular elimination machinery in cadmium detoxification.« less

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

PubMed Central

Renes, Johan; de Vries, Elisabeth G E; Nienhuis, Edith F; Jansen, Peter L M; Müller, Michael

1999-01-01

The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4′,4′-azo-n-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations ⩾10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 μM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1. PMID:10188979

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

PubMed Central

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. PMID:24244377

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

PubMed

Holland, M L; Lau, D T T; Allen, J D; Arnold, J C

2007-11-01

Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

PubMed

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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

PubMed

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

2009-02-01

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

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

PubMed Central

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

PubMed

Denecke, Shane; Fusetto, Roberto; Batterham, Philip

2017-12-01

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a Reconstituted System†

PubMed Central

Shukla, Sudhanshu; Rai, Versha; Saini, Preeti; Banerjee, Dibyendu; Menon, Anant K.; Prasad, Rajendra

2008-01-01

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that ~70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With ~6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was ~15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function. PMID:17924650

Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance transporter.

PubMed

Steiger, Scott A; Li, Chun; Backos, Donald S; Reigan, Philip; Natale, N R

2017-06-15

A series of dimeric isoxazolyl-1,4-dihydropyridines (IDHPs) were prepared by click chemistry and examined for their ability to bind the multi-drug resistance transporter (MDR-1), a member of the ATP-binding cassette superfamily (ABC). Eight compounds in the present study exhibited single digit micromolar binding to this efflux transporter. One monomeric IDHP m-Br-1c, possessed submicromolar binding of 510nM at MDR-1. Three of the dimeric IDHPs possessed <1.5µM activity, and 4b and 4c were observed to have superior binding selectivity compared to their corresponding monomers verses the voltage gated calcium channel (VGCC). The dimer with the best combination of activity and selectivity for MDR-1 was analog 4c containing an m-Br phenyl moiety in the 3-position of the isoxazole, and a tether with five ethyleneoxy units, referred to herein as Isoxaquidar. Two important controls, mono-triazole 5 and pyridine 6, also were examined, indicating that the triazole - incorporated as part of the click assembly as a spacer - contributes to MDR-1 binding. Compounds were also assayed at the allosteric site of the mGluR5 receptor, as a GPCR 7TM control, indicating that the p-Br IDHPs 4d, 4e and 4f with tethers of from n=2 to 5 ethylenedioxy units, had sub-micromolar affinities with 4d being the most efficacious at 193nM at mGluR5. The results are interpreted using a docking study using a human ABC as our current working hypothesis, and suggest that the distinct SARs emerging for these three divergent classes of biomolecular targets may be tunable, and amenable to the development of further selectivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multidrug transport protein norM from vibrio cholerae simultaneously couples to sodium- and proton-motive force.

PubMed

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W

2014-05-23

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

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

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

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells.

PubMed

Gelhaus, Stacy L; Gilad, Oren; Hwang, Wei-Ting; Penning, Trevor M; Blair, Ian A

2012-02-25

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids

PubMed Central

Holland, M L; Lau, D T T; Allen, J D; Arnold, J C

2007-01-01

Background and purpose: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. Experimental approach: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. Key results: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (−)-11-nor-9-carboxy-Δ9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. Conclusions and implications: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates. PMID:17906686

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

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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes: A historic approach and recent advances.

PubMed

Stefan, Sven Marcel; Wiese, Michael

2018-05-29

Multidrug resistance-associated protein 1 (MRP1, ABCC1) is an ATP-binding cassette (ABC) transport protein. This efflux pump uses the energy of ATP hydrolysis to export structurally diverse antineoplastic agents in human cancers. The upregulation of MRP1 (either inherent or acquired) is one major reason for the occurrence of the phenomenon called multidrug resistance (MDR). MDR is characterized by a reduced outcome of chemotherapy due to the active intracellular clearance of cytostatic drugs below the necessary effect concentration. Much effort has been made to overcome MDR, which implied high-throughput screenings of already known pharmacological and natural compounds, modification of intrinsic substrates, as well as design and synthesis of new inhibitors. This review is meant not only to summarize the most recent results over the past 10 years, but also to highlight major achievements regarding reversal of MRP1-mediated MDR, from the time of its discovery until today. The focus lies on small-molecule compounds that feature either direct MRP1 inhibition/transport blockage, toxicity against MRP1-overexpressing cells, inhibition/modification of intracellular processes necessary for MRP1 function, or modification of MRP1-related metabolic and genomic mechanisms. Considering all aspects, this review might be useful to (re)consider possible strategies to overcome MRP1-mediated MDR. Furthermore, it may be the basis for developing new, even better, highly potent, less toxic, and selective (as well as broad-spectrum) MRP1 inhibitors that will enter clinical evaluations in different malignancies and finally conduce to overcome MDR in general. © 2018 Wiley Periodicals, Inc.

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

PubMed

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

2017-08-01

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. Copyright © 2017. Published by Elsevier B.V.

Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG.

PubMed

Luo, Qingshan; Yang, Xu; Yu, Shan; Shi, Huigang; Wang, Kun; Xiao, Le; Zhu, Guangyu; Sun, Chuanqi; Li, Tingting; Li, Dianfan; Zhang, Xinzheng; Zhou, Min; Huang, Yihua

2017-05-01

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB 2 FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB 2 FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB 2 FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

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.

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

PubMed Central

Pahnke, Jens; Langer, Oliver; Krohn, Markus

2014-01-01

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

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

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

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

PubMed Central

Zhao, Jinlei

2014-01-01

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments. PMID:24957625

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

PubMed

Adebesin, Funmilayo; Widhalm, Joshua R; Boachon, Benoît; Lefèvre, François; Pierman, Baptiste; Lynch, Joseph H; Alam, Iftekhar; Junqueira, Bruna; Benke, Ryan; Ray, Shaunak; Porter, Justin A; Yanagisawa, Makoto; Wetzstein, Hazel Y; Morgan, John A; Boutry, Marc; Schuurink, Robert C; Dudareva, Natalia

2017-06-30

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

Karanjin interferes with ABCB1, ABCC1, and ABCG2.

PubMed

Michaelis, Martin; Rothweiler, Florian; Nerreter, Thomas; Sharifi, Mohsen; Ghafourian, Taravat; Cinatl, Jindrich

2014-01-01

The prominent ATP-binding cassette (ABC) transporters ABCB1, ABCC1, and ABCG2 are involved in substance transport across physiological barriers and therefore in drug absorption, distribution, and elimination. They also mediate multi-drug resistance in cancer cells. Different flavonoids are known to interfere with different ABC transporters. Here, the effect of the furanoflavonol karanjin, a potential drug with antiglycaemic, gastroprotective, antifungal, and antibacterial effects, was investigated on ABCB1, ABCC1, and ABCG2-mediated drug transport in comparison to the flavonoids apigenin, genistein, and naringenin. Cells expressing the relevant transporters (ABCB1: UKF-NB-3(ABCB1), UKF-NB-3(r)VCR¹⁰; ABCC1: G62, PC-3(r)VCR²⁰; ABCG2: UKF-NB-3(ABCG2)) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities. Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1 µM. Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs.

Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex.

PubMed

Oswald, Christine; Jenewein, Stefan; Smits, Sander H J; Holland, I Barry; Schmitt, Lutz

2008-04-01

TNP-modified nucleotides have been used extensively to study protein-nucleotide interactions. In the case of ABC-ATPases, application of these powerful tools has been greatly restricted due to the significantly higher affinity of the TNP-nucleotide for the corresponding ABC-ATPase in comparison to the non-modified nucleotides. To understand the molecular changes occurring upon binding of the TNP-nucleotide to an ABC-ATPase, we have determined the crystal structure of the TNP-ADP/HlyB-NBD complex at 1.6A resolution. Despite the higher affinity of TNP-ADP, no direct fluorophore-protein interactions were observed. Unexpectedly, only water-mediated interactions were detected between the TNP moiety and Tyr(477), that is engaged in pi-pi stacking with the adenine ring, as well as with two serine residues (Ser(504) and Ser(509)) of the Walker A motif. Interestingly, the side chains of these two serine residues adopt novel conformations that are not observed in the corresponding ADP structure. However, in the crystal structure of the S504A mutant, which binds TNP-ADP with similar affinity to the wild type enzyme, a novel TNP-water interaction compensates for the missing serine side chain. Since this water molecule is not present in the wild type enzyme, these results suggest that only water-mediated interactions provide a structural explanation for the increased affinity of TNP-nucleotides towards ABC-ATPases. However, our results also imply that in silico approaches such as docking or modeling cannot directly be applied to generate 'affinity-adopted' ADP- or ATP-analogs for ABC-ATPases.

Effect of drug efflux transporters on placental transport of antiretroviral agent abacavir.

PubMed

Neumanova, Zuzana; Cerveny, Lukas; Greenwood, Susan L; Ceckova, Martina; Staud, Frantisek

2015-11-01

Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer. Copyright © 2015 Elsevier Inc. All rights reserved.

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...

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

PubMed

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

2010-02-01

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

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

PubMed Central

Stindt, Jan; Smits, Sander H. J.; Schmitt, Lutz

2013-01-01

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters. PMID:23593265

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

PubMed Central

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

2011-01-01

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

Increasing the cytotoxicity of doxorubicin in breast cancer MCF-7 cells with multidrug resistance using a mesoporous silica nanoparticle drug delivery system.

PubMed

Wang, Xin; Teng, Zhaogang; Wang, Haiyan; Wang, Chunyan; Liu, Ying; Tang, Yuxia; Wu, Jiang; Sun, Jin; Wang, Hai; Wang, Jiandong; Lu, Guangming

2014-01-01

Resistance to cytotoxic chemotherapy is the main cause of therapeutic failure and death in women with breast cancer. Overexpression of various members of the superfamily of adenosine triphosphate binding cassette (ABC)-transporters has been shown to be associated with multidrug resistance (MDR) phenotype in breast cancer cells. MDR1 protein promotes the intracellular efflux of drugs. A novel approach to address cancer drug resistance is to take advantage of the ability of nanocarriers to sidestep drug resistance mechanisms by endosomal delivery of chemotherapeutic agents. Doxorubicin (DOX) is an anthracycline antibiotic commonly used in breast cancer chemotherapy and a substrate for ABC-mediated drug efflux. In the present study, we developed breast cancer MCF-7 cells with overexpression of MDR1 and designed mesoporous silica nanoparticles (MSNs) which were used as a drug delivery system. We tested the efficacy of DOX in the breast cancer cell line MCF-7/MDR1 and in a MCF-7/MDR1 xenograft nude mouse model using the MSNs drug delivery system. Our data show that drug resistance in the human breast cancer cell line MCF-7/MDR1 can be overcome by treatment with DOX encapsulated within mesoporous silica nanoparticles.

Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

PubMed

Nishikawa, Joy L; Boeszoermenyi, Andras; Vale-Silva, Luis A; Torelli, Riccardo; Posteraro, Brunella; Sohn, Yoo-Jin; Ji, Fei; Gelev, Vladimir; Sanglard, Dominique; Sanguinetti, Maurizio; Sadreyev, Ruslan I; Mukherjee, Goutam; Bhyravabhotla, Jayaram; Buhrlage, Sara J; Gray, Nathanael S; Wagner, Gerhard; Näär, Anders M; Arthanari, Haribabu

2016-02-25

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption

PubMed Central

Reznicek, Josef; Ceckova, Martina; Ptackova, Zuzana; Martinec, Ondrej; Tupova, Lenka; Cerveny, Lukas

2017-01-01

ABSTRACT Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo. Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir. PMID:28696229

Ketoconazole and the modulation of multidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1 drug transport models.

PubMed

Fan, Y; Rodriguez-Proteau, R

2008-02-01

The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby altering cellular uptake and apparent permeability (P(app)) of multidrug-resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2, MDCKII-MDR1, and MDCKII wild-type cell transport models. (3)H-CSA/(3)H-digoxin transport experiments were performed with and without co-exposure to ketoconazole, and (3)H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3-gallate, and xanthohumol. Ketoconazole (3 microM) reduced the P(app) efflux of CSA and digoxin from 5.07 x 10(-6) to 2.91 x 10(-6) cm s(-1) and from 2.60 x 10(-6) to 1.41 x 10(-6) cm s(-1), respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the P(app) efflux of CSA and increased the P(app) absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin, whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses. In conclusion, ketoconazole modulates the P(app) of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole.

Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

PubMed

Wiseman, Benjamin; Kilburg, Arnaud; Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

PubMed Central

Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli. PMID:25517996

HG-829 Is a Potent Noncompetitive Inhibitor of the ATP-Binding Cassette Multidrug Resistance Transporter ABCB1

PubMed Central

Caceres, Gisela; Robey, Robert W.; Sokol, Lubomir; McGraw, Kathy L.; Clark, Justine; Lawrence, Nicholas J.; Sebti, Said M.; Wiese, Michael; List, Alan F.

2015-01-01

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrugresistant malignancies. PMID:22761337

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

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

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption.

PubMed

Reznicek, Josef; Ceckova, Martina; Ptackova, Zuzana; Martinec, Ondrej; Tupova, Lenka; Cerveny, Lukas; Staud, Frantisek

2017-09-01

Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir. Copyright © 2017 American Society for Microbiology.

Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes.

PubMed

Mitterbauer, R; Heinrich, M; Rauscher, R; Lemmens, M; Bürstmayr, H; Adam, G

2003-03-01

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.

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

ABC transporter content diversity in Streptococcus pneumoniae impacts competence regulation and bacteriocin production.

PubMed

Wang, Charles Y; Patel, Nisha; Wholey, Wei-Yun; Dawid, Suzanne

2018-06-19

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) uses natural genetic competence to increase its adaptability through horizontal gene transfer. One method of acquiring DNA is through predation of neighboring strains with antimicrobial peptides called "bacteriocins." Competence and production of the major family of pneumococcal bacteriocins, pneumocins, are regulated by the quorum-sensing systems com and blp , respectively. In the classical paradigm, the ABC transporters ComAB and BlpAB each secretes its own system's signaling pheromone and in the case of BlpAB also secretes the pneumocins. While ComAB is found in all pneumococci, only 25% of strains encode an intact version of BlpAB [BlpAB(+)] while the rest do not [BlpAB(-)]. Contrary to the classical paradigm, it was previously shown that BlpAB(-) strains can activate blp through ComAB-mediated secretion of the blp pheromone during brief periods of competence. To better understand the full extent of com - blp crosstalk, we examined the contribution of each transporter to competence development and pneumocin secretion. We found that BlpAB(+) strains have a greater capacity for competence activation through BlpAB-mediated secretion of the com pheromone. Similarly, we show that ComAB and BlpAB are promiscuous and both can secrete pneumocins. Consequently, differences in pneumocin secretion between BlpAB(+) and BlpAB(-) strains derive from the regulation and kinetics of transporter expression rather than substrate specificity. We speculate that BlpAB(-) strains (opportunists) use pneumocins mainly in a narrowly tailored role for DNA acquisition and defense during competence while BlpAB(+) strains (aggressors) expand their use for the general inhibition of rival strains. Copyright © 2018 the Author(s). Published by PNAS.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells.

PubMed

Saeed, Mohamed E M; Meyer, Marion; Hussein, Ahmed; Efferth, Thomas

2016-06-20

Traditional medicine plays a major role for primary health care worldwide. Cancer belongs to the leading disease burden in industrialized and developing countries. Successful cancer therapy is hampered by the development of resistance towards established anticancer drugs. In the present study, we investigated the cytotoxicity of 29 extracts from 26 medicinal plants of South-Africa against leukemia cell lines, most of which are used traditionally to treat cancer and related symptoms. We have investigated the plant extracts for their cytotoxic activity towards drug-sensitive parental CCRF-CEM leukemia cells and their multidrug-resistant P-glycoprotein-overexpressing subline, CEM/ADR5000 by means of the resazurin assay. A panel of 60 NCI tumor cell lines have been investigated for correlations between selected phytochemicals from medicinal plants and the expression of resistance-conferring genes (ABC-transporters, oncogenes, tumor suppressor genes). Seven extracts inhibited both cell lines (Acokanthera oppositifolia, Hypoestes aristata, Laurus nobilis, Leonotis leonurus, Plectranthus barbatus, Plectranthus ciliates, Salvia apiana). CEM/ADR5000 cells exhibited a low degree of cross-resistance (3.35-fold) towards the L. leonurus extract, while no cross-resistance was observed to other plant extracts, although CEM/ADR5000 cells were highly resistant to clinically established drugs. The log10IC50 values for two out of 14 selected phytochemicals from these plants (acovenoside A and ouabain) of 60 tumor cell lines were correlated to the expression of ABC-transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS) and tumor suppressors (TP53). Sensitivity or resistance of the cell lines were not statistically associated with the expression of these genes, indicating that multidrug-resistant, refractory tumors expressing these genes may still respond to acovenoside A and ouabain. The bioactivity of South African medicinal plants may represent a basis for the development

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

PubMed Central

Wang, Zhao; Fan, Guizhen; Hryc, Corey F; Blaza, James N; Serysheva, Irina I; Schmid, Michael F; Chiu, Wah; Luisi, Ben F; Du, Dijun

2017-01-01

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump. DOI: http://dx.doi.org/10.7554/eLife.24905.001 PMID:28355133

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

PubMed

Staud, Frantisek; Cerveny, Lukas; Ceckova, Martina

2012-11-01

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

The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin.

PubMed

Menges, R; Muth, G; Wohlleben, W; Stegmann, E

2007-11-01

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

Masitinib Antagonizes ATP-Binding Cassette Subfamily C Member 10-Mediated Paclitaxel Resistance: A Preclinical Study

PubMed Central

Kathawala, Rishil J.; Sodani, Kamlesh; Chen, Kang; Patel, Atish; Abuznait, Alaa H.; Anreddy, Nagaraju; Sun, Yue-Li; Kaddoumi, Amal; Ashby, Charles R.; Chen, Zhe-Sheng

2014-01-01

Paclitaxel displays clinical activity against a wide variety of solid tumors. However, resistance to paclitaxel significantly attenuates the response to chemotherapy. The ABC transporter subfamily C member 10 (ABCC10), also known as multi-drug resistance protein 7 (MRP7) efflux transporter, is a major mediator of paclitaxel resistance. In this study, we show that masitinib, a small molecule stem-cell growth factor receptor (c-Kit) tyrosine kinase inhibitor, at non-toxic concentrations, significantly attenuates paclitaxel resistance in HEK293 cells transfected with ABCC10. Our in vitro studies indicated that masitinib (2.5 μM) enhanced the intracellular accumulation and decreased the efflux of paclitaxel by inhibiting the ABCC10 transport activity without altering the expression level of ABCC10 protein. Furthermore, masitinib, in combination with paclitaxel, significantly inhibited the growth of ABCC10-expressing tumors in nude athymic mice in vivo. Masitinib administration also resulted in a significant increase in the levels of paclitaxel in the plasma, tumors and lungs compared to paclitaxel alone. In conclusion, the combination of paclitaxel and masitinib could serve as a novel and useful therapeutic strategy to reverse paclitaxel resistance mediated by ABCC10. PMID:24431074

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

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

PubMed

Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

2014-07-29

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

PubMed

Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

2010-03-01

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

Mdr65 decreases toxicity of multiple insecticides in Drosophila melanogaster.

PubMed

Sun, Haina; Buchon, Nicolas; Scott, Jeffrey G

2017-10-01

ABC transporters are ubiquitous membrane-bound proteins, present in both prokaryotes and eukaryotes. The major function of eukaryotic ABC transporters is to mediate the efflux of a variety of substrates (including xenobiotics) out of cells. ABC transporters have been widely investigated in humans, particularly for their involvement in multidrug resistance (MDR). Considerably less is known about their roles in transport and/or excretion in insects. ABC transporters are only known to function as exporters in insects. Drosophila melanogaster has 56 ABC transporter genes, including eight which are phylogenetically most similar to the human Mdr genes (ABCB1 clade). We investigated the role of ABC transporters in the ABCB1 clade in modulating the susceptibility to insecticides. We took advantage of the GAL4/UAS system in D. melanogaster to knockdown the expression levels of Mdr65, Mdr50, Mdr49 and ABCB6 using transgenic UAS-RNAi lines and conditional driver lines. The most notable effects were increased sensitivities to nine different insecticides by silencing of Mdr65. Furthermore, a null mutation of Mdr65 decreased the malathion, malaoxon and fipronil LC 50 values by a factor of 1.9, 2.1 and 3.9, respectively. Altogether, this data demonstrates the critical role of ABC transporters, particularly Mdr65, in altering the toxicity of specific, structurally diverse, insecticides in D. melanogaster. Copyright © 2017 Elsevier Ltd. All rights reserved.

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer

PubMed Central

Hedditch, Ellen L.; Gao, Bo; Russell, Amanda J.; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E.; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T.; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W.; Ekici, Arif B.; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K.; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P.; Berchuck, Andrew; Goode, Ellen; Bowtell, David D.; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D.; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J.

2014-01-01

Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA–mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan–Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the “A” subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e−6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor

ABCA transporter gene expression and poor outcome in epithelial ovarian cancer.

PubMed

Hedditch, Ellen L; Gao, Bo; Russell, Amanda J; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W; Ekici, Arif B; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P; Berchuck, Andrew; Goode, Ellen; Bowtell, David D; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J

2014-07-01

ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. Associations with outcome were observed with ABC transporters of the "A" subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid

Iowa ABC connections : [tech transfer summary].

DOT National Transportation Integrated Search

2015-06-01

The Iowa Department of Transportation (DOT) and other organizations have : been developing accelerated bridge construction (ABC) concepts, details, and : processes, and Iowa has come to be viewed as a national leader in the area of : ABC. However, th...

Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin

PubMed Central

Chedik, Lisa; Bruyere, Arnaud; Le Vee, Marc; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Potin, Sophie; Fardel, Olivier

2017-01-01

Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 μM (OCT1 inhibition by allethrin) to 77.6 μM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 μM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient

Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane▿

PubMed Central

Hassan, Karl A.; Souhani, Talal; Skurray, Ronald A.; Brown, Melissa H.

2008-01-01

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein. PMID:18223078

Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul

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 mousemore » 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.« less

Purification of a Multidrug Resistance Transporter for Crystallization Studies

PubMed Central

Alegre, Kamela O.; Law, Christopher J.

2015-01-01

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

Molecular mechanisms of multidrug resistance in cancer chemotherapy.

PubMed

Nooter, K; Stoter, G

1996-07-01

The occurrence of multidrug resistance (MDR) is one of the main obstacles in the successful chemotherapeutic treatment of cancer. MDR cell lines are resistant to the so-called naturally occurring anti-cancer drugs, such as anthracyclines, Vinca alkaloids and epipodophyllotoxins, but are not cross-resistant to alkylating agents, antimetabolites and cisplatin. So far, three separate forms of MDR have been characterized in more detail: classical MDR, non-Pgp MDR and atypical MDR. Although all three MDR phenotypes have much in common with respect to cross-resistance patterns, the underlying mechanisms certainly differ. Atypical MDR is associated with quantitative and qualitative alterations in topoisomerase II alpha, a nuclear enzyme that actively participates in the lethal action of cytotoxic drugs. Atypical MDR cells do not overexpress P-glycoprotein, and are unaltered in their ability to accumulate drugs. In this review we will focus on classical and non-Pgp MDR. The molecular mechanism of classical and non-Pgp MDR is transcriptional activation of membrane-bound transport proteins. These transport proteins belong to the ATP-binding cassette (ABC) superfamily of transport systems. The classical MDR phenotype is characterized by a reduced ability to accumulate drugs, due to activity of an energy-dependent uni-directional, membrane-bound, drug-efflux pump with broad substrate specificity. The classical MDR drug pump is composed of a transmembrane glycoprotein (P-glyco-protein-Pgp) with a molecular weight of 170 kD, and is, in man, encoded by the so-called multidrug resistance (MDR1) gene. Typically, non-Pgp MDR has no P-gly-coprotein expression, yet has about the same cross-resistance pattern as classical MDR. This non-Pgp MDR phenotype is caused by overexpression of the multidrug resistance-associated protein (MRP) gene, which encodes a 190 kD membrane-bound glycoprotein (MRP). MRP probably works by direct extrusion of cytotoxic drugs from the cell and/or by mediating

Single photon emission computed tomography and positron emission tomography imaging of multi-drug resistant P-glycoprotein--monitoring a transport activity important in cancer, blood-brain barrier function and Alzheimer's disease.

PubMed

Piwnica-Worms, David; Kesarwala, Aparna H; Pichler, Andrea; Prior, Julie L; Sharma, Vijay

2006-11-01

Overexpression of multi-drug resistant P-glycoprotein (Pgp) remains an important barrier to successful chemotherapy in cancer patients and impacts the pharmacokinetics of many important drugs. Pgp is also expressed on the luminal surface of brain capillary endothelial cells wherein Pgp functionally comprises a major component of the blood-brain barrier by limiting central nervous system penetration of various therapeutic agents. In addition, Pgp in brain capillary endothelial cells removes amyloid-beta from the brain. Several single photon emission computed tomography and positron emission tomography radiopharmaceutical have been shown to be transported by Pgp, thereby enabling the noninvasive interrogation of Pgp-mediated transport activity in vivo. Therefore, molecular imaging of Pgp activity may enable noninvasive dynamic monitoring of multi-drug resistance in cancer, guide therapeutic choices in cancer chemotherapy, and identify transporter deficiencies of the blood-brain barrier in Alzheimer's disease.

Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter.

PubMed

Escudero, Leticia; Mariscal, Vicente; Flores, Enrique

2015-08-01

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. Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in

Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

PubMed

Martins, A; Spengler, G; Martins, M; Rodrigues, L; Viveiros, M; Davin-Regli, A; Chevalier, J; Couto, I; Pagès, J M; Amaral, L

2010-10-01

Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively. Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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

Iowa ABC connections.

DOT National Transportation Integrated Search

2015-06-01

For several years the Iowa Department of Transportation (DOT), Iowa State University, the Federal Highway Administration, : and several Iowa counties have been working to develop accelerated bridge construction (ABC) concepts, details, and processes....

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; Annoura, Takeshi; Matz, Joachim M; Kenthirapalan, Sanketha; Kooij, Taco W A; Matuschewski, Kai; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Siebelink-Stoter, Rianne; Graumans, Wouter; Ramesar, Jai; Klop, Onny; Russel, Frans G M; Sauerwein, Robert W; Janse, Chris J; Franke-Fayard, Blandine M; Koenderink, Jan B

2016-07-01

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species. © 2016 John Wiley & Sons Ltd.

Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

PubMed Central

Akan, Ilhan; Akan, Selma; Akca, Hakan; Savas, Burhan; Ozben, Tomris

2005-01-01

Background Multidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs. Results N-acetylcysteine increased the resistance of both cells against vincristine and BSO decreased NAC-enhanced MRP1-mediated vincristine resistance, indicating that induction of MRP1-mediated vincristine resistance depends on GSH. Vincristine decreased cellular GSH concentration and increased GPx activity. Glutathione S-Transferase activity was decreased by NAC. Conclusion Our results demonstrate that NAC and BSO have opposite effects in MRP1 mediated vincristine resistance and BSO seems a promising chemotherapy improving agent in MRP1 overexpressing tumor cells. PMID:16042792

ABC transporters and cytochromes P450 in the human central nervous system: influence on brain pharmacokinetics and contribution to neurodegenerative disorders.

PubMed

Dutheil, Fabien; Jacob, Aude; Dauchy, Sandrine; Beaune, Philippe; Scherrmann, Jean-Michel; Declèves, Xavier; Loriot, Marie-Anne

2010-10-01

The identification of xenobiotic metabolizing enzymes (i.e., CYP) and transporters (i.e., ABC transporters) (XMET) in the human brain, including the BBB, raises the question whether these transporters and enzymes have specific functions in brain physiology, neuropharmacology and toxicology. Relevant literature was identified using PubMed search articles published up to March 2010. Search terms included 'ABC transporters and P450 or CYP', 'drug metabolism, effect and toxicity' and 'neurodegenerative disease (Alzheimer and Parkinson diseases)' restricted to the field of 'brain or human brain'. This review aims to provide a better understanding of XMET functions in the human brain and show their pharmacological importance for improving drug delivery and efficacy and also for managing their side effects. Finally, the impact of brain XMET activity during neurodegenerative processes is discussed, giving an opportunity to identify new markers of human brain diseases. During the last 2 decades, much evidence concerning the specific distribution patterns of XMET, their induction by xenobiotics and endobiotics and their genetic variations have made cerebral ABC transporters and CYP enzymes key elements in the way individual patients respond to centrally acting drugs.

Opioid transport by ATP-binding cassette transporters at the blood-brain barrier: implications for neuropsychopharmacology.

PubMed

Tournier, Nicolas; Declèves, Xavier; Saubaméa, Bruno; Scherrmann, Jean-Michel; Cisternino, Salvatore

2011-01-01

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.

THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

PubMed Central

Su, Wendy; Pasternak, Gavril W.

2013-01-01

The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

Lack of Influence of Substrate on Ligand Interaction with the Human Multidrug and Toxin Extruder, MATE1

PubMed Central

Martínez-Guerrero, Lucy J.; Morales, Mark; Ekins, Sean

2016-01-01

Multidrug and toxin extruder (MATE) 1 plays a central role in mediating renal secretion of organic cations, a structurally diverse collection of compounds that includes ∼40% of prescribed drugs. Because inhibition of transport activity of other multidrug transporters, including the organic cation transporter (OCT) 2, is influenced by the structure of the transported substrate, the present study screened over 400 drugs as inhibitors of the MATE1-mediated transport of four structurally distinct organic cation substrates: the commonly used drugs: 1) metformin and 2) cimetidine; and two prototypic cationic substrates, 3) 1-methyl-4-phenylpyridinium (MPP), and 4) the novel fluorescent probe, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium iodide. Transport was measured in Chinese hamster ovary cells that stably expressed the human ortholog of MATE1. Comparison of the resulting inhibition profiles revealed no systematic influence of substrate structure on inhibitory efficacy. Similarly, IC50 values for 26 structurally diverse compounds revealed no significant influence of substrate structure on the kinetic interaction of inhibitor with MATE1. The IC50 data were used to generate three-dimensional quantitative pharmacophores that identified hydrophobic regions, H-bond acceptor sites, and an ionizable (cationic) feature as key determinants for ligand binding to MATE1. In summary, in contrast to the behavior observed with some other multidrug transporters, including OCT2, the results suggest that substrate identity exerts comparatively little influence on ligand interaction with MATE1. PMID:27418674

Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter

USDA-ARS?s Scientific Manuscript database

The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against multiple pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whet...

Anticancer Effects of the Nitric Oxide-Modified Saquinavir Derivative Saquinavir-NO against Multidrug-Resistant Cancer Cells12

PubMed Central

Rothweiler, Florian; Michaelis, Martin; Brauer, Peter; Otte, Jürgen; Weber, Kristoffer; Fehse, Boris; Doerr, Hans Wilhelm; Wiese, Michael; Kreuter, Jörg; Al-Abed, Yousef; Nicoletti, Ferdinando; Cinatl, Jindrich

2010-01-01

The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors. PMID:21170266

Multidrug Efflux Transporters Limit Accumulation of Inorganic, but Not Organic, Mercury in Sea Urchin Embryos

PubMed Central

Bošnjak, Ivana; Uhlinger, Kevin R.; Heim, Wesley; Smital, Tvrtko; Franekić-Čolić, Jasna; Coale, Kenneth; Epel, David; Hamdoun, Amro

2011-01-01

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 (HgCl2) and organic (CH3HgCl) 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. PMID:19924972

Multi-Drug Resistance Transporter 2 Regulates Mucosal Inflammation by Facilitating the Synthesis of Hepoxilin A3

PubMed Central

Pazos, Michael; Siccardi, Dario; Mumy, Karen L.; Bien, Jeffrey D.; Louie, Steve; Shi, Hai Ning; Gronert, Karsten; Mrsny, Randall J.; McCormick, Beth A.

2008-01-01

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A3, an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotatic gradient to guide neutrophils from the submucosa, across epithelia to the luminal site of an inflammatory stimulus - the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A3 is secreted from the intestinal epithelium during an inflammatory insult. In this study we reveal that hepoxilin A3 is a substrate for the apical efflux ABC transporter, multi-drug resistance protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A3 synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A3 synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions. PMID:19017997

Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages

PubMed Central

Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

2014-01-01

Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to

NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.

PubMed

Ducos, Eric; Fraysse, Staffan; Boutry, Marc

2005-12-19

In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family.

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

PubMed Central

Wei, Shipeng; Roessler, Bryan C.; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L.; Kirk, Kevin L.

2014-01-01

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. PMID:24876383

Virtual screening of ABCC1 transporter nucleotidebinding domains as a therapeutic target in multidrug resistant cancer

PubMed Central

Rungsardthong, Kanin; Mares- Sámano, Sergio; Penny, Jeffrey

2012-01-01

ABCC1 is a member of the ATP-binding Cassette super family of transporters, actively effluxes xenobiotics from cells. Clinically, ABCC1 expression is linked to cancer multidrug resistance. Substrate efflux is energised by ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) and inhibition of these events may help combat drug resistance. The aim of this study is to identify potential inhibitors of ABCC1 through virtual screening of National Cancer Institute (NCI) compounds. A threedimensional model of ABCC1 NBD2 was generated using MODELLER whilst the X-ray crystal structure of ABCC1 NBD1 was retrieved from the Protein Data Bank. A pharmacophore hypothesis was generated based on flavonoids known to bind at the NBDs using PHASE, and used to screen the NCI database. GLIDE was employed in molecular docking studies for all hit compounds identified by pharmacophore screening. The best potential inhibitors were identified as compounds possessing predicted binding affinities greater than ATP. Approximately 5% (13/265) of the hit compounds possessed lower docking scores than ATP in ABCC1 NBD1 (NSC93033, NSC662377, NSC319661, NSC333748, NSC683893, NSC226639, NSC94231, NSC55979, NSC169121, NSC166574, NSC73380, NSC127738, NSC115534), whereas approximately 7% (7/104) of docked NCI compounds were predicted to possess lower docking scores than ATP in ABCC1 NBD2 (NSC91789, NSC529483, NSC211168, NSC318214, NSC116519, NSC372332, NSC526974). Analyses of docking orientations revealed P-loop residues of each NBD and the aromatic amino acids Trp653 (NBD1) and Tyr1302 (NBD2) were key in interacting with high-affinity compounds. On the basis of docked orientation and docking score the compounds identified may be potential inhibitors of ABCC1 and require further pharmacological analysis. Abbreviations ABC - ATP-binding cassette, DHS - dehydrosilybin, MDR - multidrug resistance, NBD - nucleotide-binding domain, PDB - protein data bank. PMID:23144549

Cross-site randomized control trial of the Social ABCs caregiver-mediated intervention for toddlers with autism spectrum disorder.

PubMed

Brian, Jessica A; Smith, Isabel M; Zwaigenbaum, Lonnie; Bryson, Susan E

2017-10-01

To evaluate the efficacy of the Social ABCs parent-mediated intervention for toddlers with suspected or confirmed autism spectrum disorder (ASD), through a cross-site randomized control trial, sixty-three parent-toddler dyads (toddler age: 16-30 months) were randomized into treatment (Social ABCs) or control (service-as-usual) conditions. Video data were obtained at three key time-points: Baseline; Post-training (PT; week 12); and Follow-Up (week 24). Analyses included 62 dyads. Treatment allocation significantly accounted for PT gains, all favouring the Treatment group, in (1) child functional vocal responsiveness to parent prompts (R 2  = 0.43, P < .001), (2) child vocal initiations (R 2  = 0.28, P < .001), (3) parent smiling (R 2  = 0.09, P = .017), and (4) fidelity of implementation (R 2  = 0.71, P < .001). A trend was observed for increased social orienting (R 2  = 0.06, P = 0.054); gains in parent smiling significantly predicted increases in child smiling and social orienting. Parents in the treatment condition reported significant gains in self-efficacy following the intervention (P = 0.009). No differential effects emerged for performance on standardized measures. The Social ABCs is a relatively low-resource, efficacious intervention, with potential to be a cost-effective means of intervening at the first signs of possible ASD. Autism Res 2017, 10: 1700-1711. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. Clinical Trial Title: Social ABCs for Toddlers with Signs of Autism Spectrum Disorder: RCT of a Parent-Mediated Intervention http//ClinicalTrials.gov identifier: NCT02428452. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

NFκBP65 transcription factor modulates resistance to doxorubicin through ABC transporters in breast cancer.

PubMed

Velaei, Kobra; Samadi, Nasser; Soltani, Sina; Barazvan, Balal; Soleimani Rad, Jafar

2017-07-01

Shedding light on chemoresistance biology of breast cancer could contribute to enhance the clinical outcome. Intrinsic or acquired resistance to chemotherapy is a major problem in breast cancer treatment. The NFκB pathway by siRNAP65 and JSH-23 as a translocational inhibitor of NFκBP65 in the doxorubicin-resistant MCF-7 (MCF-7/Dox) and MCF-7 cells was blocked. Then, the ABC transporter expression and function were assessed by real-time qRT-PCR and flow cytometry, respectively. Induction of apoptosis was evaluated after inhibition of the NFΚB pathway as well. Our study underlined the upregulation of NFκBP65 and anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax in the MCF-7/Dox cells compared with control MCF-7 cells. Here, we showed that interplay between nuclear factor kappa B P65 (NFkBP65) as a transcriptional regulator and ABC transporters in the MCF-7/Dox cancer cells. We found that inhibition of the elevated expression of NFκBP65 in the resistant breast cancer, whether translocational inhibition or silencing by siRNA, decreased the expression and function of MDR1 and MRP1 efflux pumps. Furthermore, the blockade of NFκBP65 promoted apoptosis via modulating Bcl-2 and BAX expression. After inhibition of the NFκBP65 signaling pathway, elevated baseline expression of survival Bcl-2 gene in the resistant breast cells significantly decreased. Suppression of the NFκB pathway has a profound dual impact on promoting the intrinsic apoptotic pathway and reducing ABC transporter function and expression, which are some of the chemoresistance features. It was speculated that the NFκB pathway directly acts on doxorubicin-induced MDR1 and MRP1 expression in MCF-7/Dox cells.

Inhibition of Glutathione Peroxidase Mediates the Collateral Sensitivity of Multidrug-resistant Cells to Tiopronin*

PubMed Central

Hall, Matthew D.; Marshall, Travis S.; Kwit, Alexandra D. T.; Miller Jenkins, Lisa M.; Dulcey, Andrés E.; Madigan, James P.; Pluchino, Kristen M.; Goldsborough, Andrew S.; Brimacombe, Kyle R.; Griffiths, Gary L.; Gottesman, Michael M.

2014-01-01

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic. PMID:24930045

Effect of COPD treatments on MRP1-mediated transport in bronchial epithelial cells

PubMed Central

van der Deen, Margaretha; Homan, Sandra; Timmer-Bosscha, Hetty; Scheper, Rik J; Timens, Wim; Postma, Dirkje S; de Vries, Elisabeth G

2008-01-01

Background Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD. Methods The immortalized human bronchial epithelial cell line 16HBE14o− was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry. Results Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner. Conclusions Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD. PMID:18990976

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

PubMed

Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

2008-02-26

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

Piperine, a piperidine alkaloid from Piper nigrum re-sensitizes P-gp, MRP1 and BCRP dependent multidrug resistant cancer cells.

PubMed

Li, Sen; Lei, Yu; Jia, Yingjie; Li, Na; Wink, Michael; Ma, Yonggang

2011-12-15

Over-expression of P-gp, MRP1 and BCRP in tumor cells is one of the important mechanisms leading to multidrug resistance (MDR), which impairs the efficacy of chemotherapy. P-gp, MRP1 and BCRP are ABC (ATP-Binding Cassette) transporters, which can expel a variety of lipophilic anti-cancer drugs and protect tumor cells. During a screening of MDR reversal agents among alkaloids of various structural types, a piperidine alkaloid, piperine (a main piperidine alkaloid in Piper nigurm) was identified as an inhibitor. Piperine can potentiate the cytotoxicity of anti-cancer drugs in resistant sublines, such as MCF-7/DOX and A-549/DDP, which were derived from MCF-7 and A-549 cell lines. At a concentration of 50 μM piperine could reverse the resistance to doxorubicin 32.16 and 14.14 folds, respectively. It also re-sensitized cells to mitoxantrone 6.98 folds. In addition, long-term treatment of cells by piperine inhibits transcription of the corresponding ABC transporter genes. These results suggest that piperine can reverse MDR by multiple mechanisms and it may be a promising lead compound for future studies. Copyright © 2011 Elsevier GmbH. All rights reserved.

A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

PubMed

Manzano, J I; García-Hernández, R; Castanys, S; Gamarro, F

2013-08-01

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.

Basic Residues R260 and K357 Affect the Conformational Dynamics of the Major Facilitator Superfamily Multidrug Transporter LmrP

PubMed Central

Wang, Wei; van Veen, Hendrik W.

2012-01-01

Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H+(Na+) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

PubMed

Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

2011-01-25

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae.

PubMed

Shahi, Puja; Gulshan, Kailash; Näär, Anders M; Moye-Rowley, W Scott

2010-07-15

The multiprotein transcriptional Mediator complex provides a key link between RNA polymerase II and upstream transcriptional activator proteins. Previous work has established that the multidrug resistance transcription factors Pdr1 and Pdr3 interact with the Mediator component Med15/Gal11 to drive normal levels of expression of the ATP-binding cassette transporter-encoding gene PDR5 in Saccharomyces cerevisiae. PDR5 transcription is induced upon loss of the mitochondrial genome (rho(0) cells) and here we provide evidence that this rho(0) induction is Med15 independent. A search through other known Mediator components determined that Med12/Srb8, a member of the CDK8 Mediator submodule, is required for rho(0) activation of PDR5 transcription. The CDK8 submodule contains the cyclin C homologue (CycC/Srb11), cyclin-dependent kinase Cdk8/Srb10, and the large Med13/Srb9 protein. Loss of these other proteins did not lead to the same block in PDR5 induction. Chromatin immunoprecipitation analyses demonstrated that Med15 is associated with the PDR5 promoter in both rho(+) and rho(0), whereas Med12 recruitment to this target promoter is highly responsive to loss of the mitochondrial genome. Coimmunoprecipitation experiments revealed that association of Pdr3 with Med12 can only be detected in rho(0) cells. These experiments uncover the unique importance of Med12 in activated transcription of PDR5 seen in rho(0) cells.

Icotinib antagonizes ABCG2-mediated multidrug resistance, but not the pemetrexed resistance mediated by thymidylate synthase and ABCG2

PubMed Central

Shukla, Suneet; Zhang, Yun-Kai; Wang, Yi-Jun; Kathawala, Rishil J.; Robey, Robert W.; Zhang, Li; Yang, Dong-Hua; Talele, Tanaji T.; Bates, Susan E.; Ambudkar, Suresh V.; Chen, Zhe-Sheng

2014-01-01

ABCG2 is a potential biomarker causing multidrug resistance (MDR) in Non-Small Cell Lung Cancer (NSCLC). We conducted this study to investigate whether Icotinib, a small-molecule inhibitor of EGFR tyrosine kinase, could interact with ABCG2 transporter in NSCLC. Our results showed that Icotinib reversed ABCG2-mediated MDR by antagonizing the drug efflux function of ABCG2. Icotinib stimulated the ATPase activity in a concentration-dependent manner and inhibited the photolabeling of ABCG2 with [125I]-Iodoarylazidoprazosin, demonstrating that it interacts at the drug-binding pocket. Homology modeling predicted the binding conformation of Icotinib at Asn629 centroid-based grid of ABCG2. However, Icotinib at reversal concentration did not affect the expression levels of AKT and ABCG2. Furthermore, a combination of Icotinib and topotecan exhibited significant synergistic anticancer activity against NCI-H460/MX20 tumor xenografts. However, the inhibition of transport activity of ABCG2 was insufficient to overcome pemetrexed resistance in NCI-H460/MX20 cells, which was due to the co-upregulated thymidylate synthase (TS) and ABCG2 expression. This is the first report to show that the up-regulation of TS in ABCG2-overexpressing cell line NCI-H460/MX20 may play a role of resistance to pemetrexate. Our findings suggested different possible strategies of overcoming the resistance of topotecan and pemetrexed in the NSCLC patients. PMID:24980828

Icotinib antagonizes ABCG2-mediated multidrug resistance, but not the pemetrexed resistance mediated by thymidylate synthase and ABCG2.

PubMed

Wang, De-Shen; Patel, Atish; Shukla, Suneet; Zhang, Yun-Kai; Wang, Yi-Jun; Kathawala, Rishil J; Robey, Robert W; Zhang, Li; Yang, Dong-Hua; Talele, Tanaji T; Bates, Susan E; Ambudkar, Suresh V; Xu, Rui-Hua; Chen, Zhe-Sheng

2014-06-30

ABCG2 is a potential biomarker causing multidrug resistance (MDR) in Non-Small Cell Lung Cancer (NSCLC). We conducted this study to investigate whether Icotinib, a small-molecule inhibitor of EGFR tyrosine kinase, could interact with ABCG2 transporter in NSCLC. Our results showed that Icotinib reversed ABCG2-mediated MDR by antagonizing the drug efflux function of ABCG2. Icotinib stimulated the ATPase activity in a concentration-dependent manner and inhibited the photolabeling of ABCG2 with [125I]-Iodoarylazidoprazosin, demonstrating that it interacts at the drug-binding pocket. Homology modeling predicted the binding conformation of Icotinib at Asn629 centroid-based grid of ABCG2. However, Icotinib at reversal concentration did not affect the expression levels of AKT and ABCG2. Furthermore, a combination of Icotinib and topotecan exhibited significant synergistic anticancer activity against NCI-H460/MX20 tumor xenografts. However, the inhibition of transport activity of ABCG2 was insufficient to overcome pemetrexed resistance in NCI-H460/MX20 cells, which was due to the co-upregulated thymidylate synthase (TS) and ABCG2 expression. This is the first report to show that the up-regulation of TS in ABCG2-overexpressing cell line NCI-H460/MX20 may play a role of resistance to pemetrexate. Our findings suggested different possible strategies of overcoming the resistance of topotecan and pemetrexed in the NSCLC patients.

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

The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury.

PubMed

Tribull, Tiffany E; Bruner, Richard H; Bain, Lisa J

2003-04-30

We examined the ability of the multidrug resistance-associated protein 1 (MRP1/ABCC1) to transport pesticides, as this transporter mediates the cellular efflux of a variety of xenobiotics, typically as glucuronide, sulfate, or glutathione conjugates. NIH3T3 cells stably expressing MRP1 were 3.37-fold more resistant to the toxicity of fenitrothion, 3.12-fold more resistant to chlorpropham, and 2.5-fold more resistant to methoxychlor, a pesticide with estrogenic and anti-androgenic metabolites. The cells expressing MRP1 also eliminated methoxychlor two times more rapidly than their mock-transfected counterparts. We then examined whether mrp1 expression could alter the toxicity of methoxychlor in vivo using male FVB/mrp1 knockout mice (FVB/mrp1-/-). Both control and knockout mice were fed 25 mg/kg methoxychlor in honey for 39 days, and its effects on testicular morphology were examined. Methoxychlor treatment did not significantly affect testicular morphology in the FVB mice, but markedly reduced the number of developing spermatocytes in the FVB/mrp1-/- mice. These results suggest that MRPI may play a role in protecting the seminiferous tubules from methoxychlor-induced damage.

The ABC protein turned chloride channel whose failure causes cystic fibrosis

NASA Astrophysics Data System (ADS)

Gadsby, David C.; Vergani, Paola; Csanády, László

2006-03-01

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos.

PubMed

Bošnjak, Ivana; Pleić, Ivana Lepen; Borra, Marco; Mladineo, Ivona

2013-12-01

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2.

PubMed

Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

2015-01-01

Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic.

A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

PubMed

Khun, H H; Kirby, S D; Lee, B C

1998-05-01

The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

A Neisseria meningitidis fbpABC Mutant Is Incapable of Using Nonheme Iron for Growth

PubMed Central

Khun, Heng H.; Kirby, Shane D.; Lee, B. Craig

1998-01-01

The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway. PMID:9573125

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

PubMed

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

... DEPARTMENT OF TRANSPORTATION Surface Transportation Board [Docket No. FD 35397] ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D..., ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, Massachusetts (STB...

Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

PubMed

Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I

2013-01-01

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

Independent Activity of the Homologous Small Regulatory RNAs AbcR1 and AbcR2 in the Legume Symbiont Sinorhizobium meliloti

PubMed Central

Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I.

2013-01-01

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia. PMID:23869210

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

PubMed

Steglich, Matthias; Hofmann, Julia D; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

2018-01-01

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p -cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile , with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile , we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production

PubMed Central

Steglich, Matthias; Hofmann, Julia D.; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

2018-01-01

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291. PMID:29867812

ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

PubMed Central

Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

2015-01-01

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

Polymorphisms in ABC Transporter Genes and Concentrations of Mercury in Newborns – Evidence from Two Mediterranean Birth Cohorts

PubMed Central

Llop, Sabrina; Engström, Karin; Ballester, Ferran; Franforte, Elisa; Alhamdow, Ayman; Pisa, Federica; Tratnik, Janja Snoj; Mazej, Datja; Murcia, Mario; Rebagliato, Marisa; Bustamante, Mariona; Sunyer, Jordi; Sofianou-Katsoulis, Αikaterini; Prasouli, Alexia; Antonopoulou, Eleni; Antoniadou, Ioanna; Nakou, Sheena; Barbone, Fabio; Horvat, Milena; Broberg, Karin

2014-01-01

Background The genetic background may influence methylmercury (MeHg) metabolism and neurotoxicity. ATP binding cassette (ABC) transporters actively transport various xenobiotics across biological membranes. Objective To investigate the role of ABC polymorphisms as modifiers of prenatal exposure to MeHg. Methods The study population consisted of participants (n = 1651) in two birth cohorts, one in Italy and Greece (PHIME) and the other in Spain (INMA). Women were recruited during pregnancy in Italy and Spain, and during the perinatal period in Greece. Total mercury concentrations were measured in cord blood samples by atomic absorption spectrometry. Maternal fish intake during pregnancy was determined from questionnaires. Polymorphisms (n = 5) in the ABC genes ABCA1, ABCB1, ABCC1 and ABCC2 were analysed in both cohorts. Results ABCB1 rs2032582, ABCC1 rs11075290, and ABCC2 rs2273697 modified the associations between maternal fish intake and cord blood mercury concentrations. The overall interaction coefficient between rs2032582 and log2-transformed fish intake was negative for carriers of GT (β = −0.29, 95%CI −0.47, −0.12) and TT (β = −0.49, 95%CI −0.71, −0.26) versus GG, meaning that for a doubling in fish intake of the mothers, children with the rs2032582 GG genotype accumulated 35% more mercury than children with TT. For rs11075290, the interaction coefficient was negative for carriers of TC (β = −0.12, 95%CI −0.33, 0.09), and TT (β = −0.28, 95%CI −0.51, −0.06) versus CC. For rs2273697, the interaction coefficient was positive when combining GA+AA (β = 0.16, 95%CI 0.01, 0.32) versus GG. Conclusion The ABC transporters appear to play a role in accumulation of MeHg during early development. PMID:24831289

The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

PubMed

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-06-17

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter.

PubMed

Underwood, William; Somerville, Shauna C

2017-10-03

The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3.

Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

PubMed

Liao, Zheng-Gen; Tang, Tao; Guan, Xue-Jing; Dong, Wei; Zhang, Jing; Zhao, Guo-Wei; Yang, Ming; Liang, Xin-Li

2016-11-24

P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.

2014-10-02

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate.more » The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.« less

Overcoming Multidrug Resistance through the GLUT1-Mediated and Enzyme-Triggered Mitochondrial Targeting Conjugate with Redox-Sensitive Paclitaxel Release.

PubMed

Ma, Pengkai; Chen, Jianhua; Bi, Xinning; Li, Zhihui; Gao, Xing; Li, Hongpin; Zhu, Hongyu; Huang, Yunfang; Qi, Jing; Zhang, Yujie

2018-04-18

Multidrug resistance (MDR) is thought to be the major obstacle leading to the failure of paclitaxel (PTX) chemotherapy. To solve this problem, a glucose transporter-mediated and matrix metalloproteinase 2 (MMP2)-triggered mitochondrion-targeting conjugate [glucose-polyethylene glycol (PEG)-peptide-triphenylphosponium-polyamidoamine (PAMAM)-PTX] composed of a PAMAM dendrimer and enzymatic detachable glucose-PEG was constructed for mitochondrial delivery of PTX. The conjugate was characterized by a 30 nm sphere particle, MMP2-sensitive PEG outer layer detachment from PAMAM, and glutathione (GSH)-sensitive PTX release. It showed higher cellular uptake both in glucose transporter 1 (GLUT1) overexpressing MCF-7/MDR monolayer cell (2D) and multicellular tumor spheroids (3D). The subcellular location study showed that it could specifically accumulate in the mitochondria. Moreover, it exhibited higher cytotoxicity against MCF-7/MDR cells, which significantly reverse the MDR of MCF-7/MDR cells. The MDR reverse might be caused by reducing the ATP content through destroying the mitochondrial membrane as well as by down-regulating P-gp expression. In vivo imaging and tissue distribution indicated more conjugate accumulated in the tumor of the tumor-bearing mice model. Consequently, the conjugate showed better tumor inhibition rate and lower body weight loss, which demonstrated that it possessed high efficiency and low toxicity. This study provides glucose-mediated GLUT targeting, MMP2-responsive PEG detachment, triphenylphosponium-mediated mitochondria targeting, and a GSH-sensitive intracellular drug release conjugate that has the potential to be exploited for overcoming MDR of PTX.

Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans.

PubMed Central

Broeks, A; Gerrard, B; Allikmets, R; Dean, M; Plasterk, R H

1996-01-01

Acquired resistance of mammalian cells to multiple chemotherapeutic drugs can result from enhanced expression of the multidrug resistance-associated protein (MRP), which belongs to the ABC transporter superfamily. ABC transporters play a role in the protection of organisms against exogenous toxins by cellular detoxification processes. We have identified four MRP homologues in the soil nematode Caenorhabditis elegans, and we have studied one member, mrp-1, in detail. Using an mrp::lacZ gene fusion, mrp-l expression was found in cells of the pharynx, the pharynx-intestinal valve and the anterior intestinal cells, the rectum-intestinal valve and the epithelial cells of the vulva. Targeted inactivation of mrp-l resulted in increased sensitivity to the heavy metal ions cadmium and arsenite, to which wild-type worms are highly tolerant. The most pronounced effect of the mrp-1 mutation is on the ability of animals to recover from temporary exposure to high concentrations of heavy metals. Nematodes were found to be hypersensitive to heavy metals when both the MRP homologue, mrp-1, and a member of the P-glycoprotein (Pgp) gene family, pgp-1, were deleted. We conclude that nematodes have multiple proteins, homologues of mammalian proteins involved in the cellular resistance to chemotherapeutic drugs, that protect them against heavy metals. Images PMID:8947035

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...

Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs☆

PubMed Central

Martinelli, Axel; Henriques, Gisela; Cravo, Pedro; Hunt, Paul

2011-01-01

In malaria parasites, mutations in two genes of folate biosynthesis encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) modify responses to antifolate therapies which target these enzymes. However, the involvement of other genes which modify the availability of exogenous folate, for example, has been proposed. Here, we used short-read whole-genome re-sequencing to determine the mutations in a clone of the rodent malaria parasite, Plasmodium chabaudi, which has altered susceptibility to both sulphadoxine and pyrimethamine. This clone bears a previously identified S106N mutation in dhfr and no mutation in dhps. Instead, three additional point mutations in genes on chromosomes 2, 13 and 14 were identified. The mutated gene on chromosome 13 (mdr2 K392Q) encodes an ABC transporter. Because Quantitative Trait Locus analysis previously indicated an association of genetic markers on chromosome 13 with responses to individual and combined antifolates, MDR2 is proposed to modulate antifolate responses, possibly mediated by the transport of folate intermediates. PMID:20858498

ATP-Binding Cassette Efflux Transporters in Human Placenta

PubMed Central

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Interaction of Isoflavones with the BCRP/ABCG2 Drug Transporter

PubMed Central

Bircsak, Kristin M; Aleksunes, Lauren M

2015-01-01

This review will provide a comprehensive overview of the interactions between dietary isoflavones and the ATP-binding cassette (ABC) G2 efflux transporter, which is also named the breast cancer resistance protein (BCRP). Expressed in a variety of organs including the liver, kidneys, intestine, and placenta, BCRP mediates the disposition and excretion of numerous endogenous chemicals and xenobiotics. Isoflavones are a class of naturally-occurring compounds that are found at high concentrations in commonly consumed foods and dietary supplements. A number of isoflavones, including genistein and daidzein and their metabolites, interact with BCRP as substrates, inhibitors, and/or modulators of gene expression. To date, a variety of model systems have been employed to study the ability of isoflavones to serve as substrates and inhibitors of BCRP; these include whole cells, inverted plasma membrane vesicles, in situ organ perfusion, as well as in vivo rodent and sheep models. Evidence suggests that BCRP plays a role in mediating the disposition of isoflavones and in particular, their conjugated forms. Furthermore, as inhibitors, these compounds may aid in reversing multidrug resistance and sensitizing cancer cells to chemotherapeutic drugs. This review will also highlight the consequences of altered BCRP expression and/or function on the pharmacokinetics and toxicity of chemicals following isoflavone exposure. PMID:26179608

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

PubMed

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

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

Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques.

PubMed

Wong, Kelvin; Briddon, Stephen J; Holliday, Nicholas D; Kerr, Ian D

2016-01-01

ABCG2 is one of three human ATP binding cassette (ABC) transporters involved in the export from cells of a chemically and structurally diverse range of compounds. This multidrug efflux capability, together with a broad tissue distribution in the body, means that ABCG2 exerts a range of effects on normal physiology such as kidney urate transport, as well as contributing towards the pharmacokinetic profiles of many exogenous drugs. The primary sequence of ABCG2 contains only half the number of domains required for a functioning ABC transporter and so it must oligomerise in order to function, yet its oligomeric state in intact cell membranes remains uncharacterized. We have analysed ABCG2 in living cell membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, and stepwise photobleaching to demonstrate a predominantly tetrameric structure for ABCG2 in the presence or absence of transport substrates. These results provide the essential basis for exploring pharmacological manipulation of oligomeric state as a strategy to modulate ABCG2 activity in future selective therapeutics. Copyright © 2015 Elsevier B.V. All rights reserved.

Hijacking membrane transporters for arsenic phytoextraction

PubMed Central

LeBlanc, Melissa S.; McKinney, Elizabeth C.; Meagher, Richard B.; Smith, Aaron P.

2012-01-01

Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator. PMID:23108027

Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin

2017-08-01

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A directmore » observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.« less

ATP-binding cassette exporters: structure and mechanism with a focus on P-glycoprotein and MRP1.

PubMed

Arana, Maite Rocío; Altenberg, Guillermo

2017-10-12

The majority of proteins that belong to the ATP-binding cassette (ABC) superfamily are transporters that mediate the efflux of substrates from cells. These exporters include multidrug resistance proteins of the ABCB and ABCC subfamilies, such as P-glycoprotein (Pgp) and MRP1, respectively. These proteins are not only involved in the resistance of cancer to cytotoxic agents, but also in the protection from endo and xenobiotics, and the determination of drug pharmacokinetics, as well as in the pathophysiology of a variety of disorders. Here, we present a review of the information available on ABC exporters, with a focus on Pgp, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

PubMed Central

KATHAWALA, RISHIL J.; CHEN, JUN-JIANG; ZHANG, YUN-KAI; WANG, YI-JUN; PATEL, ATISH; WANG, DE-SHEN; TALELE, TANAJI T.; ASHBY, CHARLES R.; CHEN, ZHE-SHENG

2014-01-01

In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 μM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 μM) significantly increased the intracellular accumulation of [3H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [3H]-MX. However, masitinib (2.5 μM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression. PMID:24626598

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

PubMed Central

Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

2005-01-01

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

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

... DEPARTMENT OF TRANSPORTATION Surface Transportation Board [STB Finance Docket No. 35356] 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 under 49 CFR 1150.31 to lease from O...

Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.

PubMed

Haider, Ameena J; Cox, Megan H; Jones, Natalie; Goode, Alice J; Bridge, Katherine S; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D

2015-07-17

ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. © 2015 Authors.

Screening dietary flavonoids for the reversal of P-glycoprotein-mediated multidrug resistance in cancer

PubMed Central

Mohana, S; Ganesan, M; Agilan, B; Karthikeyan, R; Srithar, G; Beaulah Mary, R; Ananthakrishnan, D; Velmurugan, D; Rajendra Prasad, N; Ambudkar, Suresh V.

2016-01-01

P-glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance in cancer cells. Although various approaches of virtual screening procedures have been practiced so far to develop first three generations of P-gp inhibitors, their toxicity and drug interaction profiles are still a matter of concern. To address the above important problem of developing safe and effective P-gp inhibitors, we have made systematic computational and experimental studies on the interaction of natural phytochemicals with human P-gp. Molecular docking and QSAR studies were carried out for 40 dietary phytochemicals in the drug-binding site of the transmembrane domains (TMDs) of P-gp. Dietary flavonoids exhibit better interactions with homology modeled human P-gp. Based on the computational analysis, selected flavonoids were tested for their inhibitory potential against P-gp transport function in drug resistant cell lines using calcein-AM and rhodamine 123 efflux assays. It has been found that quercetin and rutin were the highly desirable flavonoids for the inhibition of P-gp transport function and significantly reduced resistance in cytotoxicity assay to paclitaxel in P-gp overexpressing MDR cell lines. Hence, quercetin and rutin may be considered as potential chemosensitizing agents to overcome multidrug resistance in cancer. PMID:27216424

Screening dietary flavonoids for the reversal of P-glycoprotein-mediated multidrug resistance in cancer.

PubMed

Mohana, S; Ganesan, M; Agilan, B; Karthikeyan, R; Srithar, G; Beaulah Mary, R; Ananthakrishnan, D; Velmurugan, D; Rajendra Prasad, N; Ambudkar, Suresh V

2016-07-19

P-Glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance in cancer cells. Although various screening procedures have been practiced so far to develop first three generations of P-gp inhibitors, their toxicity and drug interaction profiles are still a matter of concern. To address the above important problem of developing safe and effective P-gp inhibitors, we have made systematic computational and experimental studies on the interaction of natural phytochemicals with human P-gp. Molecular docking and QSAR studies were carried out for 40 dietary phytochemicals in the drug-binding site of the transmembrane domains (TMDs) of P-gp. Dietary flavonoids exhibit better interactions with homology modeled human P-gp. Based on the computational analysis, selected flavonoids were tested for their inhibitory potential against P-gp transport function in drug resistant cell lines using calcein-AM and rhodamine 123 efflux assays. It has been found that quercetin and rutin were the highly desirable flavonoids for the inhibition of P-gp transport function and they significantly reduced resistance in cytotoxicity assays to paclitaxel in P-gp overexpressing MDR cell lines. Hence, quercetin and rutin may be considered as potential chemosensitizing agents to overcome multidrug resistance in cancer.

ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells.

PubMed

Hinrichs, John W J; Klappe, Karin; Hummel, Ina; Kok, Jan W

2004-02-13

In this study we show that P-glycoprotein in multidrug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multidrug-resistant HT29col human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched membrane domains. This localization is independent of caveolae, since 2780AD cells do not express caveolin-1. Although HT29col cells do express caveolin-1, the ATP-binding cassette transporter and caveolin-1 were dissociated on the basis of differential solubility in Triton X-100 and absence of microscopical colocalization. While both the multidrug resistance-associated protein 1 and caveolin-1 are located in Lubrol-based membrane domains, they occupy different regions of these domains.

Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury.

PubMed

Lee, Hyunjung; McKeon, Robert J; Bellamkonda, Ravi V

2010-02-23

Chondroitin sulfate proteoglycans (CSPGs) are a major class of axon growth inhibitors that are up-regulated after spinal cord injury (SCI) and contribute to regenerative failure. Chondroitinase ABC (chABC) digests glycosaminoglycan chains on CSPGs and can thereby overcome CSPG-mediated inhibition. But chABC loses its enzymatic activity rapidly at 37 degrees C, necessitating the use of repeated injections or local infusions for a period of days to weeks. These infusion systems are invasive, infection-prone, and clinically problematic. To overcome this limitation, we have thermostabilized chABC and developed a system for its sustained local delivery in vivo, obviating the need for chronically implanted catheters and pumps. Thermostabilized chABC remained active at 37 degrees C in vitro for up to 4 weeks. CSPG levels remained low in vivo up to 6 weeks post-SCI when thermostabilized chABC was delivered by a hydrogel-microtube scaffold system. Axonal growth and functional recovery following the sustained local release of thermostabilized chABC versus a single treatment of unstabilized chABC demonstrated significant differences in CSPG digestion. Animals treated with thermostabilized chABC in combination with sustained neurotrophin-3 delivery showed significant improvement in locomotor function and enhanced growth of cholera toxin B subunit-positive sensory axons and sprouting of serotonergic fibers. Therefore, improving chABC thermostability facilitates minimally invasive, sustained, local delivery of chABC that is potentially effective in overcoming CSPG-mediated regenerative failure. Combination therapy with thermostabilized chABC with neurotrophic factors enhances axonal regrowth, sprouting, and functional recovery after SCI.

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

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

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

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 LptB 2 FG 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 LptB 2 FG 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. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

Neratinib reverses ATP-binding cassette B1-mediated chemotherapeutic drug resistance in vitro, in vivo, and ex vivo.

PubMed

Zhao, Xiao-qin; Xie, Jing-dun; Chen, Xing-gui; Sim, Hong May; Zhang, Xu; Liang, Yong-ju; Singh, Satyakam; Talele, Tanaji T; Sun, Yueli; Ambudkar, Suresh V; Chen, Zhe-Sheng; Fu, Li-wu

2012-07-01

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Neratinib Reverses ATP-Binding Cassette B1-Mediated Chemotherapeutic Drug Resistance In Vitro, In Vivo, and Ex Vivo

PubMed Central

Zhao, Xiao-qin; Xie, Jing-dun; Chen, Xing-gui; Sim, Hong May; Zhang, Xu; Liang, Yong-ju; Singh, Satyakam; Talele, Tanaji T.; Sun, Yueli; Ambudkar, Suresh V.; Chen, Zhe-Sheng

2012-01-01

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [125I]iodoarylazidoprazosin in a concentration-dependent manner (IC50 = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function. PMID:22491935

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

PubMed

Jonsson, Ing-Marie; Juuti, Jarmo T; François, Patrice; AlMajidi, Rana; Pietiäinen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J; Driessen, Arnold J M; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P

2010-12-02

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

PubMed

Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

2015-03-30

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

NASA Astrophysics Data System (ADS)

Song, Xianfeng; Setayeshgar, Sima; Cole, Bryan; Hamdoun, Amro; Epel, David

2008-03-01

Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology 276 452 (2004).

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

PubMed

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

2015-07-01

Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake

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

Minireview: SLCO and ABC Transporters: A Role for Steroid Transport in Prostate Cancer Progression

PubMed Central

Cho, Eunpi; Montgomery, R. Bruce

2014-01-01

Androgens play a critical role in the development and progression of prostate cancer (PCa), and androgen deprivation therapy via surgical or medical castration is front-line therapy for patients with advanced PCa. However, intratumoral testosterone levels are elevated in metastases from patients with castration-resistant disease, and residual intratumoral androgens have been implicated in mediating ligand-dependent mechanisms of androgen receptor activation. The source of residual tissue androgens present despite castration has not been fully elucidated, but proposed mechanisms include uptake and conversion of adrenal androgens, such as dehdroepiandrosterone to testosterone and dihydrotestosterone, or de novo androgen synthesis from cholesterol or progesterone precursors. In this minireview, we discuss the emerging evidence that suggests a role for specific transporters in mediating transport of steroids into or out of prostate cells, thereby influencing intratumoral androgen levels and PCa development and progression. We focus on the solute carrier and ATP binding cassette gene families, which have the most published data for a role in PCa-related steroid transport, and review the potential impact of genetic variation on steroid transport activity and PCa outcomes. Continued assessment of transport activity in PCa models and human tumor tissue is needed to better delineate the different roles these transporters play in physiologic and neoplastic settings, and in order to determine whether targeting the uptake of steroid substrates by specific transporters may be a clinically feasible therapeutic strategy. PMID:25147980

Uremic Toxins Inhibit Transport by Breast Cancer Resistance Protein and Multidrug Resistance Protein 4 at Clinically Relevant Concentrations

PubMed Central

Mutsaers, Henricus A. M.; van den Heuvel, Lambertus P.; Ringens, Lauke H. J.; Dankers, Anita C. A.; Russel, Frans G. M.; Wetzels, Jack F. M.; Hoenderop, Joost G.; Masereeuw, Rosalinde

2011-01-01

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [3H]-methotrexate ([3H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [3H]-estrone sulfate ([3H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [3H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC50 value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations. PMID:21483698

Bypassing multidrug resistance in human breast cancer cells with lipid/polymer particle assemblies

PubMed Central

Li, Bo; Xu, Hui; Li, Zhen; Yao, Mingfei; Xie, Meng; Shen, Haijun; Shen, Song; Wang, Xinshi; Jin, Yi

2012-01-01

Background Multidrug resistance (MDR) mediated by the overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), remains one of the major obstacles to effective cancer chemotherapy. In this study, lipid/particle assemblies named LipoParticles (LNPs), consisting of a dimethyldidodecylammonium bromide (DMAB)-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticle core surrounded by a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) shell, were specially designed for anticancer drugs to bypass MDR in human breast cancer cells that overexpress P-gp. Methods Doxorubicin (DOX), a chemotherapy drug that is a P-gp substrate, was conjugated to PLGA and encapsulated in the self-assembled LNP structure. Physiochemical properties of the DOX-loaded LNPs were characterized in vitro. Cellular uptake, intracellular accumulation, and cytotoxicity were compared in parental Michigan Cancer Foundation (MCF)-7 cells and P-gp-overexpressing, resistant MCF-7/adriamycin (MCF-7/ADR) cells. Results This study found that the DOX formulated in LNPs showed a significantly increased accumulation in the nuclei of drug-resistant cells relative to the free drug, indicating that LNPs could alter intracellular traffic and bypass drug efflux. The cytotoxicity of DOX loaded-LNPs had a 30-fold lower half maximal inhibitory concentration (IC50) value than free DOX in MCF-7/ADR, measured by the colorimetric cell viability (MTT) assay, correlated with the strong nuclear retention of the drug. Conclusion The results show that this core-shell lipid/particle structure could be a promising strategy to bypass MDR. PMID:22275834

The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2.

PubMed

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

2007-12-01

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 analogue 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 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). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 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 with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show 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.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

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

PubMed

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

2016-02-01

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

Regulation of Expression of abcA and Its Response to Environmental Conditions

PubMed Central

Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

2014-01-01

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates*

PubMed Central

Fredslund, Folmer; Vujičić Žagar, Andreja; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan

2016-01-01

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

PubMed

Ghanem, Carolina I; Rudraiah, Swetha; Bataille, Amy M; Vigo, María B; Goedken, Michael J; Manautou, José E

2015-04-01

Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention. Copyright © 2015 Elsevier Inc. All rights reserved.

An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

PubMed Central

Urban, M; Bhargava, T; Hamer, J E

1999-01-01

Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms. PMID:9927411

Multidrug resistance in enteric and other gram-negative bacteria.

PubMed

George, A M

1996-05-15

In Gram-negative bacteria, multidrug resistance is a term that is used to describe mechanisms of resistance by chromosomal genes that are activated by induction or mutation caused by the stress of exposure to antibiotics in natural and clinical environments. Unlike plasmid-borne resistance genes, there is no alteration or degradation of drugs or need for genetic transfer. Exposure to a single drug leads to cross-resistance to many other structurally and functionally unrelated drugs. The only mechanism identified for multidrug resistance in bacteria is drug efflux by membrane transporters, even though many of these transporters remain to be identified. The enteric bacteria exhibit mostly complex multidrug resistance systems which are often regulated by operons or regulons. The purpose of this review is to survey molecular mechanisms of multidrug resistance in enteric and other Gram-negative bacteria, and to speculate on the origins and natural physiological functions of the genes involved.

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux.

PubMed

Ligaba, Ayalew; Maron, Lyza; Shaff, Jon; Kochian, Leon; Piñeros, Miguel

2012-07-01

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

Maltose Uptake by the Novel ABC Transport System MusEFGK2I Causes Increased Expression of ptsG in Corynebacterium glutamicum

PubMed Central

Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W.; Krämer, Reinhard

2013-01-01

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 [14C]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. PMID:23543710

Jatrophane diterpenoids from Euphorbia sororia as potent modulators against P-glycoprotein-based multidrug resistance.

PubMed

Hu, Rui; Gao, Jie; Rozimamat, Rushangul; Aisa, Haji Akber

2018-02-25

Five new (1-5) and ten known (6-15) jatrophane diterpenoids were isolated from the fructus of Euphorbia sororia and their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis. Cytotoxicity and anti-multidrug resistance effects of these jatrophane diterpenoids were evaluated in multidrug-resistant MCF-7/ADR breast cancer cells with an overexpression of P-glycoprotein (P-gp). Eight compounds (1, 2, 4, 6, 8, 10, 11, and 15) showed promising chemoreversal abilities compared to verapamil (VRP). The most potent compound, Euphosorophane A (1), possessed many advantages, including (1) high potency (EC 50  = 92.68 ± 18.28 nM) in reversing P-gp-mediated resistance to doxorubicin (DOX), low cytotoxicity, and a high therapeutic index, (2) potency in reversing resistance to other cytotoxic agents associated with MDR, and (3) inhibition of P-gp-mediated Rhodamine123 (Rh123) efflux function in MCF-7/ADR cells. The results of the Western blot analysis indicated that the multidrug resistance (MDR) reversal induced by 1 was not due to the inhibiton of P-gp expression. Compound 1 stimulated P-gp-ATPase activity and caused the dose-dependent inhibition of DOX transport activity. Lineweaver-Burk and Dixon plots implied that 1 was a competitive inhibitor to DOX in the binding site of P-gp with a Ki of 0.49-0.50 μM. Our data suggested that 1 had a high binding affinity toward the DOX recognition site of P-gp. This resulted in inhibiting DOX transport, increasing intracellular DOX concentration, and finally resensitizing MCF-7/ADR to DOX. In addition, we discussed some added contents in the structure-activity relationship (SAR) of jatrophane diterpenoids. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

PubMed Central

Finkenwirth, Friedrich; Kirsch, Franziska

2013-01-01

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

Cross-functioning between the extraneuronal monoamine transporter and multidrug resistance protein 1 in the uptake of adrenaline and export of 5-(glutathion-S-yl)adrenaline in rat cardiomyocytes.

PubMed

Costa, Vera Marisa; Ferreira, Lusa Maria; Branco, Paula Srio; Carvalho, Flix; Bastos, Maria Lourdes; Carvalho, Rui Albuquerque; Carvalho, Mrcia; Remio, Fernando

2009-01-01

Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

Profiling of ABC transporters ABCB5, ABCF2 and nestin-positive stem cells in nevi, in situ and invasive melanoma.

PubMed

Setia, Namrata; Abbas, Ossama; Sousa, Yessica; Garb, Jane L; Mahalingam, Meera

2012-08-01

Distinct ABCB5 forms and ABCF2, members of the ATP-binding cassette (ABC) superfamily of transporters, are normally expressed in various tissues and cells, and enhanced expression of both has been demonstrated in select cancers. In melanoma cell lines, gene expression profiling of ABC transporters has revealed enhanced expression of melanocyte-specific ABCB5 and ABCF2 proteins. Given this, our primary aim was to ascertain immunohistochemical expression of the ABC transporters ABCB5 and ABCF2 and, the stem cell marker, nestin in a spectrum of benign and malignant nevomelanocytic proliferations, including nevi (n=30), in situ (n=31) and invasive (n=24) primary cutaneous melanomas to assess their role in the stepwise development of malignancy. In addition, their expression was compared with established melanoma prognosticators to ascertain their utility as independent prognosticators. A semiquantitative scoring system was utilized by deriving a cumulative score (based on percentage positivity cells and intensity of expression) and statistical analyses was carried out using analysis of variance with linear contrasts. Mean cumulative score in nevi, in situ and invasive melanoma were as follows: 3.8, 4.4 and 5.3 for ABCB5, respectively (P<0.005 for all), and 4.6, 4.6 and 5.3 for nestin, respectively (P=not significant for all). No appreciable expression of ABCF2 was noted in any of the groups. While ulcerated lesions of melanoma demonstrated lower levels of expression of ABCB5 and nestin than non-ulcerated lesions, and nestin expression was lower in lesions with mitoses >1, after controlling for the presence of ulceration and mitotic activity, the expression of both proteins did not significantly correlate with known melanoma prognosticators. The gradual increase in the expression of ABCB5 from benign nevus to in situ to invasive melanoma suggests that it plays a role in melanomagenesis. On the basis of our findings, a prospective study with follow-up data is required to

Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Němcová-Fürstová, Vlasta, E-mail: vlasta.furstova@

Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100 nM and 300 nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublinesmore » of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells. - Highlights: • Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed. • SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes. • Some taxanes are able to overcome developed resistance to

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.

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): evasion of MRP-2 mediated efflux.

PubMed

Jain, Ritesh; Agarwal, Sheetal; Mandava, Nanda Kishore; Sheng, Ye; Mitra, Ashim K

2008-10-01

Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.

Plant Flavonoids—Biosynthesis, Transport and Involvement in Stress Responses

PubMed Central

Petrussa, Elisa; Braidot, Enrico; Zancani, Marco; Peresson, Carlo; Bertolini, Alberto; Patui, Sonia; Vianello, Angelo

2013-01-01

This paper aims at analysing the synthesis of flavonoids, their import and export in plant cell compartments, as well as their involvement in the response to stress, with particular reference to grapevine (Vitis vinifera L.). A multidrug and toxic compound extrusion (MATE) as well as ABC transporters have been demonstrated in the tonoplast of grape berry, where they perform a flavonoid transport. The involvement of a glutathione S-transferase (GST) gene has also been inferred. Recently, a putative flavonoid carrier, similar to mammalian bilitranslocase (BTL), has been identified in both grape berry skin and pulp. In skin the pattern of BTL expression increases from véraison to harvest, while in the pulp its expression reaches the maximum at the early ripening stage. Moreover, the presence of BTL in vascular bundles suggests its participation in long distance transport of flavonoids. In addition, the presence of a vesicular trafficking in plants responsible for flavonoid transport is discussed. Finally, the involvement of flavonoids in the response to stress is described. PMID:23867610

Transporter-mediated biofuel secretion.

PubMed

Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

2013-05-07

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

Transporter-mediated biofuel secretion

PubMed Central

Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

2013-01-01

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as “plug-and-play” biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance. PMID:23613592

Kinetic Analysis of Rhodamines Efflux Mediated by the Multidrug Resistance Protein (MRP1)

PubMed Central

Saengkhae, Chantarawan; Loetchutinat, Chatchanok; Garnier-Suillerot, Arlette

2003-01-01

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, ka = VM/km, was very similar for the four rhodamine analogs but ∼10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes. PMID:12944313

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs

PubMed Central

Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Bagami, Mohammed Al; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael

2016-01-01

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients. PMID:27081035

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

PubMed

Fouquet, Grégory; Debuysscher, Véronique; Ouled-Haddou, Hakim; Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Al Bagami, Mohammed; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael; Marcq, Ingrid; Bouhlal, Hicham

2016-05-31

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein.

PubMed

Muñoz-Martínez, Francisco; Reyes, Carolina P; Pérez-Lomas, Antonio L; Jiménez, Ignacio A; Gamarro, Francisco; Castanys, Santiago

2006-01-01

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.

PubMed

Romsicki, Y; Sharom, F J

2001-06-12

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.

Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future.

PubMed

Cole, Susan P C

2014-01-01

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

PubMed

Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

2015-07-01

Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

PubMed Central

Aleksunes, Lauren M.

2010-01-01

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory

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.

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

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis.

PubMed

Larsen, Paul B; Geisler, Matt J B; Jones, Carol A; Williams, Kelly M; Cancel, Jesse D

2005-02-01

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

Interaction of forskolin with the P-glycoprotein multidrug transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ming s, D.I.; Seamon, K.B.; Speicher, L.A.

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 labelingmore » 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.« less

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

PubMed Central

Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

2012-01-01

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment

PubMed Central

Condic-Jurkic, Karmen; Subramanian, Nandhitha; Mark, Alan E.

2018-01-01

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment.

PubMed

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

2018-01-01

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the

The feoABC Locus of Yersinia pestis Likely Has Two Promoters Causing Unique Iron Regulation

PubMed Central

O'Connor, Lauren; Fetherston, Jacqueline D.; Perry, Robert D.

2017-01-01

The FeoABC ferrous transporter is a wide-spread bacterial system. While the feoABC locus is regulated by a number of factors in the bacteria studied, we have previously found that regulation of feoABC in Yersinia pestis appears to be unique. None of the non-iron responsive transcriptional regulators that control expression of feoABC in other bacteria do so in Y. pestis. Another unique factor is the iron and Fur regulation of the Y. pestis feoABC locus occurs during microaerobic but not aerobic growth. Here we show that this unique iron-regulation is not due to a unique aspect of the Y. pestis Fur protein but to DNA sequences that regulate transcription. We have used truncations, alterations, and deletions of the feoA::lacZ reporter to assess the mechanism behind the failure of iron to repress transcription under aerobic conditions. These studies plus EMSAs and DNA sequence analysis have led to our proposal that the feoABC locus has two promoters: an upstream P1 promoter whose expression is relatively iron-independent but repressed under microaerobic conditions and the known downstream Fur-regulated P2 promoter. In addition, we have identified two regions that bind Y. pestis protein(s), although we have not identified these protein(s) or their function. Finally we used iron uptake assays to demonstrate that both FeoABC and YfeABCD transport ferrous iron in an energy-dependent manner and also use ferric iron as a substrate for uptake. PMID:28785546

Inhibition of multidrug resistance protein 1 (MRP1) improves chemotherapy drug response in primary and recurrent glioblastoma multiforme.

PubMed

Tivnan, Amanda; Zakaria, Zaitun; O'Leary, Caitrín; Kögel, Donat; Pokorny, Jenny L; Sarkaria, Jann N; Prehn, Jochen H M

2015-01-01

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer with extremely poor prognostic outcome despite intensive treatment. All chemotherapeutic agents currently used have no greater than 30-40% response rate, many fall into the range of 10-20%, with delivery across the blood brain barrier (BBB) or chemoresistance contributing to the extremely poor outcomes despite treatment. Increased expression of the multidrug resistance protein 1(MRP1) in high grade glioma, and it's role in BBB active transport, highlights this member of the ABC transporter family as a target for improving drug responses in GBM. In this study we show that small molecule inhibitors and gene silencing of MRP1 had a significant effect on GBM cell response to temozolomide (150 μM), vincristine (100 nM), and etoposide (2 μM). Pre-treatment with Reversan (inhibitor of MRP1 and P-glycoprotein) led to a significantly improved response to cell death in the presence of all three chemotherapeutics, in both primary and recurrent GBM cells. The presence of MK571 (inhibitor of MRP1 and multidrug resistance protein 4 (MRP4) led to an enhanced effect of vincristine and etoposide in reducing cell viability over a 72 h period. Specific MRP1 inhibition led to a significant increase in vincristine and etoposide-induced cell death in all three cell lines assessed. Treatment with MK571, or specific MRP1 knockdown, did not have any effect on temozolomide drug response in these cells. These findings have significant implications in providing researchers an opportunity to improve currently used chemotherapeutics for the initial treatment of primary GBM, and improved treatment for recurrent GBM patients.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

PubMed

Murota, Yoshitaka; Tabu, Kouichi; Taga, Tetsuya

2016-11-04

Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of

Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tocchetti, Guillermo Nicolás

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a groupmore » of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs. - Highlights: • Intestinal MRP2 (ABCC2) expression and activity can be regulated by xenobiotics. • PXR and CAR are major MRP2 modulators through a transcriptional mechanism. �

Overcoming Multidrug Resistance via Photodestruction of ABCG2-Rich Extracellular Vesicles Sequestering Photosensitive Chemotherapeutics

PubMed Central

Goler-Baron, Vicky; Assaraf, Yehuda G.

2012-01-01

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing

Reversal of P-glycoprotein-mediated multidrug resistance by 5,6,7,3',4'-pentamethoxyflavone (Sinensetin).

PubMed

Choi, Cheol Hee; Sun, Kyung Hoon; An, Chun San; Yoo, Jin Cheol; Hahm, Kyung Soo; Lee, In Hwa; Sohng, Jae Kyung; Kim, Youn Chul

2002-07-26

Multidrug resistance (MDR) cells can be sensitized to anticancer drugs when treated concomitantly with chemosensitizers. In this study, chemosensitizing effects of 5,6,7,3',4'-pentamethoxyflavone (sinensetin) and its analogs were investigated with respect to in vitro efficacy and structure-activity relationship. Sinensetin reversed the resistance of P-glycoprotein (Pgp)-overexpressing AML-2/D100 to vincristine in a concentration-dependent manner. Chemosensitizing effect of sinensetin was 10- and 18-fold higher than those of 5,7,3',4'-tetramethoxyflavone and 3,7-dihydroxy-3',4'-dimethoxyflavone, respectively. Sinensetin cytotoxicity in AML-2/D100 was not changed by the complete inhibition of Pgp, suggesting that it is not a substrate for Pgp. Flow cytometry showed that sinensetin increased drug accumulation in the AML-2/D100 in a concentration-dependent manner. Unlike verapamil and cyclosporin A, the maximum non-cytotoxic concentrations of sinensetin were found to decrease the Pgp levels. Azidopine-binding assay showed that cyclosporin A or verapamil inhibited azidopine binding on Pgp partially but sinensetin did not. Taken together, these results suggest that sinensetin has a chemosensitizing effect in reversing Pgp-mediated MDR by increasing the intracellular accumulation of drugs without competition in a binding site of azidopine. Thus, sinensetin is anticipated as a novel and highly potent second-generation flavonoid chemosensitizer, since sinensetin has significant advantages of having a high therapeutic index, of being a non-transportable inhibitor, and of effecting no induction of Pgp.

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides

PubMed Central

Zibara, Kazem; Zein, Nabil El; Sabra, Mirna; Hneino, Mohammad; Harati, Hayat; Mohamed, Wael; Kobeissy, Firas H.; Kassem, Nouhad

2017-01-01

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T4 protein transporters, steady-state extraction of 125I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “L” system and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the

[Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

PubMed

Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

2013-05-04

To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

Diagnosis of cancer multidrug resistance by bacterium-mediated imaging.

PubMed

Elkadi, Omar Anwar; Abdelbasset, Muhammad

2016-04-01

Multidrug resistance (MDR) is a phenomenon expressed by many tumors affecting the chemotherapy efficacy, treatment decision, and the disease prognosis. Considering its great implication, non-invasive approaches are needed to identify this phenomenon in early stages of the disease. This article discusses the potential of the emerging non-invasive bacterium-mediated imaging of cancer in diagnosis of MDR. This potential is derived from the effect of cancer MDR on the pharmacokinetics of certain antibiotics, which are substrates of the MDR proteins. Since MDR proteins actively pump their substrates outside the resistant cancer cells, the elimination of the employed reporter bacteria, proliferating within MDR cancer cells, would require a larger dose of these antibiotics compared to those inside non-MDR cancer cells. These bacteria bear reporter genes that produce specific signals such as bioluminescent, fluorescent, magnetic, or radioactive signals that can be detected by non-invasive imaging modalities. Therefore, the presence, degree, and mechanism of MDR can be estimated by comparing the concentration of the employed antibiotic, required to cease these signals (reflecting the elimination of the bacteria), to a pre-determined reference. The real time imaging of MDR cancer and the early diagnosis of MDR, offered by this approach, would provide a better tool for preclinical studies of MDR, and allow a prompt choice of the most appropriate therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

PubMed

Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

2014-03-01

Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Genome-wide identification, phylogenetic analysis, and expression profiles of ATP-binding cassette transporter genes in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

PubMed

Xiao, Lin-Fan; Zhang, Wei; Jing, Tian-Xing; Zhang, Meng-Yi; Miao, Ze-Qing; Wei, Dan-Dan; Yuan, Guo-Rui; Wang, Jin-Jun

2018-03-01

The ATP-binding cassette (ABC) is the largest transporter gene family and the genes play key roles in xenobiotic resistance, metabolism, and development of all phyla. However, the specific functions of ABC gene families in insects is unclear. We report a genome-wide identification, phylogenetic, and transcriptional analysis of the ABC genes in the oriental fruit fly, Bactrocera dorsalis (Hendel). We identified a total of 47 ABC genes (BdABCs) from the transcriptomic and genomic databases of B. dorsalis and classified these genes into eight subfamilies (A-H), including 7 ABCAs, 7 ABCBs, 9 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 15 ABCGs, and 3 ABCHs. Comparative phylogenetic analysis of the ABCs suggests an orthologous relationship between B. dorsalis and other insect species in which these genes have been related to pesticide resistance and essential biological processes. Comparison of transcriptome and relative expression patterns of BdABCs indicated diverse multifunctions within different B. dorsalis tissues. The expression of 4, 10, and 14 BdABCs from 18 BdABCs was significantly upregulated after exposure to LD 50 s of malathion, avermectin, and beta-cypermethrin, respectively. The maximum expression level of most BdABCs (including BdABCFs, BdABCGs, and BdABCHs) occurred at 48h post exposures, whereas BdABCEs peaked at 24h after treatment. Furthermore, RNA interference-mediated suppression of BdABCB7 resulted in increased toxicity of malathion against B. dorsalis. These data suggest that ABC transporter genes might play key roles in xenobiotic metabolism and biosynthesis in B. dorsalis. Copyright © 2017 Elsevier Inc. All rights reserved.

Accelerated bridge construction (ABC) decision making and economic modeling tool.

DOT National Transportation Integrated Search

2011-12-01

In this FHWA-sponsored pool funded study, a set of decision making tools, based on the Analytic Hierarchy Process (AHP) was developed. This tool set is prepared for transportation specialists and decision-makers to determine if ABC is more effective ...

Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3/Moat-D) Is Expressed in the SAE Squalus acanthias Shark Embryo–Derived Cell Line

PubMed Central

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2008-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E. PMID:18284333

Multidrug resistance-associated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark embryo-derived cell line.

PubMed

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2007-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E.

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…

Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

PubMed

Nishino, Kunihiko

2018-01-01

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein.

PubMed

Tanaka, Yoshiki; Iwaki, Shigehiro; Tsukazaki, Tomoya

2017-09-05

The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. Copyright © 2017 Elsevier Ltd. All rights reserved.

Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters

PubMed Central

Mi, Yan-jun; Liang, Yong-ju; Huang, Hong-bing; Zhao, Hong-yun; Wu, Chung-Pu; Wang, Fang; Tao, Li-yang; Zhang, Chuan-zhao; Dai, Chun-Ling; Tiwari, Amit K.; Ma, Xiao-xu; Wah To, Kenneth Kin; Ambudkar, Suresh V.; Chen, Zhe-Sheng; Fu, Li-wu

2010-01-01

Apatinib, a small-molecule multi-targeted tyrosine kinase inhibitor, is in phase III clinical trial for treatment of patients with non-small cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr and HEK293/ABCB1 cells overexpressing ABCB1 and S1-M1-80, MCF-7/FLV1000 and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photolabeling of both ABCB1 and ABCG2 with [125I]-iodoarylazidoprazosin in a concentration-dependent fashion. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration the produced a reversal of MDRl, did not significantly alter the expression of the ABCB1 or ABCG2 protein or mRNA levels or the phosphorylation of AKT and ERK1/2. Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1 resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs. PMID:20876799

ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

PubMed

Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

2015-08-01

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice.

PubMed

Bobrov, Alexander G; Kirillina, Olga; Fosso, Marina Y; Fetherston, Jacqueline D; Miller, M Clarke; VanCleave, Tiva T; Burlison, Joseph A; Arnold, William K; Lawrenz, Matthew B; Garneau-Tsodikova, Sylvie; Perry, Robert D

2017-06-21

A number of bacterial pathogens require the ZnuABC Zinc (Zn 2+ ) transporter and/or a second Zn 2+ transport system to overcome Zn 2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn 2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn 2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn 2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn 2+ in vitro under the conditions tested. However, we detect a significant increase in Zn 2+ -binding ability of filtered supernatants from a Ybt + strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

USDA-ARS?s Scientific Manuscript database

Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

Interaction and Transport of Methamphetamine and its Primary Metabolites by Organic Cation and Multidrug and Toxin Extrusion Transporters.

PubMed

Wagner, David J; Sager, Jennifer E; Duan, Haichuan; Isoherranen, Nina; Wang, Joanne

2017-07-01

Methamphetamine is one of the most abused illicit drugs with roughly 1.2 million users in the United States alone. A large portion of methamphetamine and its metabolites is eliminated by the kidney with renal clearance larger than glomerular filtration clearance. Yet the mechanism of active renal secretion is poorly understood. The goals of this study were to characterize the interaction of methamphetamine and its major metabolites with organic cation transporters (OCTs) and multidrug and toxin extrusion (MATE) transporters and to identify the major transporters involved in the disposition of methamphetamine and its major metabolites, amphetamine and para -hydroxymethamphetamine ( p -OHMA). We used cell lines stably expressing relevant transporters to show that methamphetamine and its metabolites inhibit human OCTs 1-3 (hOCT1-3) and hMATE1/2-K with the greatest potencies against hOCT1 and hOCT2. Methamphetamine and amphetamine are substrates of hOCT2, hMATE1, and hMATE2-K, but not hOCT1 and hOCT3. p -OHMA is transported by hOCT1-3 and hMATE1, but not hMATE2-K. In contrast, organic anion transporters 1 and 3 do not interact with or transport these compounds. Methamphetamine and its metabolites exhibited complex interactions with hOCT1 and hOCT2, suggesting the existence of multiple binding sites. Our studies suggest the involvement of the renal OCT2/MATE pathway in tubular secretion of methamphetamine and its major metabolites and the potential of drug-drug interactions with substrates or inhibitors of the OCTs. This information may be considered when prescribing medications to suspected or known abusers of methamphetamine to mitigate the risk of increased toxicity or reduced therapeutic efficacy. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

EPA Science Inventory

Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

PubMed

Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

2017-11-01

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

Unstirred Water Layers and the Kinetics of Organic Cation Transport

PubMed Central

Shibayama, Takahiro; Morales, Mark; Zhang, Xiaohong; Martinez, Lucy; Berteloot, Alfred; Secomb, Timothy W.; Wright, Stephen H.

2015-01-01

Purpose Unstirred water layers (UWLs) present an unavoidable complication in the measurement of transport kinetics in cultured cells and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. Methods Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. Results Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. Conclusions Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the affinity of multidrug transporters for substrates. PMID:25791216

ABC-transporters are localized in caveolin-1-positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes.

PubMed

Ismair, Manfred G; Häusler, Stephanie; Stuermer, Claudia A; Guyot, Christelle; Meier, Peter J; Roth, Jürgen; Stieger, Bruno

2009-05-01

The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

PubMed Central

Liu, Yuan; Xu, Xiaofang; Guan, Sumin; Wu, Junzheng; Liu, Yanpu

2013-01-01

Background Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. Methods Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. Results MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. Conclusions Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors. PMID:24013781

Molecular and structural characteristics of multidrug resistance-associated protein 7 in Chinese liver fluke Clonorchis sinensis.

PubMed

Dai, Fuhong; Yoo, Won Gi; Lee, Ji-Yun; Lu, Yanyan; Pak, Jhang Ho; Sohn, Woon-Mok; Hong, Sung-Jong

2017-03-01

Multidrug resistance-associated protein 7 (MRP7, ABCC10) is a C subfamily member of the ATP-binding cassette (ABC) superfamily. MRP7 is a lipophilic anion transporter that pumps endogenous and xenobiotic substrates from the cytoplasm to the extracellular milieu. Here, we cloned and characterized CsMRP7 as a novel ABC transporter from the Chinese liver fluke, Clonorchis sinensis. Full-length cDNA of CsMRP7 was 5174 nt, encoded 1636 amino acids (aa), and harbored a 147-bp 5'-untranslated region (5'-UTR) and 116-bp 3'-UTR. Phylogenetic analysis confirmed that CsMRP7 was closer to the ABCC subfamily than the ABCB subfamily. Tertiary structures of the N-terminal region (1-322 aa) and core region (323-1621 aa) of CsMRP7 were generated by homology modeling using glucagon receptor (PDB ID: 5ee7_A) and P-glycoprotein (PDB ID: 4f4c_A) as templates, respectively. CsMRP7 nucleotide-binding domain 2 (NBD2) was conserved more than NBD1, which was the sites of ATP binding and hydrolysis. Like typical long MRPs, CsMRP7 has an additional membrane-spanning domain 0 (MSD0) and cytoplasmic loop, along with a common structural fold consisting of MSD1-NBD1-MSD2-NBD2 as a single polypeptide assembly. MSD0, MSD1, and MSD2 consisted of TM1-7, TM8-13, and TM14-19, respectively. The CsMRP7 transcript was more abundant in the metacercariae than in the adult worms. Truncated NBD1 (39 kDa) and NBD2 (44 kDa) were produced in bacteria and mouse immune sera were raised. CsMRP7 was localized in the apical side of the intestinal epithelium, sperm in the testes and seminal receptacle, receptacle membrane, and mesenchymal tissue around intestine in the adult worm. These results provide molecular information and insights into structural and functional characteristics of CsMRP7 and homologs of flukes.

Multi-Modal Strategies for Overcoming Tumor Drug Resistance: Hypoxia, Warburg’s Effect, Stem Cells, and Multifunctional Nanotechnology

PubMed Central

Milane, Lara; Ganesh, Shanthi; Shah, Shruti; Duan, Zhen-feng; Amiji, Mansoor

2011-01-01

Inefficiency in systemic drug delivery and tumor residence as well microenvironmental selection pressures contribute to the development of multidrug resistance (MDR) in cancer. Characteristics of MDR include abnormal vasculature, regions of hypoxia, up-regulation of ABC-transporters, aerobic glycolysis, and an elevated apoptotic threshold. Nano-sized delivery vehicles are ideal for treating MDR cancer as they can improve the therapeutic index of drugs and they can be engineered to achieve multifunctional parameters. The multifunctional ability of nanocarriers makes them more adept at treating heterogeneous tumor mass than traditional chemotherapy. Nanocarriers also have preferential tumor accumulation via the EPR effect; this accumulation can be further enhanced by actively targeting the biological profile of MDR cells. Perhaps the most significant benefit of using nanocarrier drug delivery to treat MDR cancer is that nanocarrier delivery diverts the effects of ABC-transporter mediated drug efflux; which is the primary mechanism of MDR. This review discusses the capabilities, applications, and examples of multifunctional nanocarriers for the treatment of MDR. This review emphasizes multifunctional nanocarriers that enhance drug delivery efficiency, the application of RNAi, modulation of the tumor apoptotic threshold, and physical approaches to overcome MDR. PMID:21497176

Signal-mediated nuclear transport in the amoeba.

PubMed

Feldherr, C M; Akin, D

1999-06-01

The evolutionary changes that occur in signal-mediated nuclear transport would be expected to reflect an increasing need to regulate nucleocytoplasmic exchanges as the complexity of organisms increases. This could involve changes in both the composition and structure of the pore complex, as well as the cytosolic factors that mediate transport. In this regard, we investigated the transport process in amoebae (Amoeba proteus and Chaos carolinensis), primitive cells that would be expected to have less stringent regulatory requirements than more complex organisms. Colloidal gold particles, coated with bovine serum albumin (BSA) conjugated with simple (large T) nuclear localization signals (NLSs), bipartite (nucleoplasmin) NLSs or mutant NLSs, were used to assay nuclear import. It was found that in amoebae (1) the diameter of the particles that are able to enter the nucleoplasm is significantly less than in vertebrate cells, (2) the simple NLS is more effective in mediating nuclear import than the bipartite NLS, and (3) the nucleoporins do not appear to be glycosylated. Evidence was also obtained suggesting that, in amoebae, the simple NLS can mediate nuclear export.

The jABC Approach to Rigorous Collaborative Development of SCM Applications

NASA Astrophysics Data System (ADS)

Hörmann, Martina; Margaria, Tiziana; Mender, Thomas; Nagel, Ralf; Steffen, Bernhard; Trinh, Hong

Our approach to the model-driven collaborative design of IKEA's P3 Delivery Management Process uses the jABC [9] for model driven mediation and choreography to complement a RUP-based (Rational Unified Process) development process. jABC is a framework for service development based on Lightweight Process Coordination. Users (product developers and system/software designers) easily develop services and applications by composing reusable building-blocks into (flow-) graph structures that can be animated, analyzed, simulated, verified, executed, and compiled. This way of handling the collaborative design of complex embedded systems has proven to be effective and adequate for the cooperation of non-programmers and non-technical people, which is the focus of this contribution, and it is now being rolled out in the operative practice.

The ABC and AUSSAT.

ERIC Educational Resources Information Center

McGarritty, Ian

1985-01-01

Discusses the Australian Broadcasting Corporation's (ABC) utilization of the AUSSAT telecommunications satellite to extend its television and radio transmission range to reach remote Australian audiences; the satellite's program gathering and interchange capabilities; and ABC's generation of other benefits to offset cost of satellite services.…

Absorption characteristic of paeoniflorin-6'-O-benzene sulfonate (CP-25) in in situ single-pass intestinal perfusion in rats.

PubMed

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

2016-09-01

1. Paeoniflorin-6'-O-benzene sulfonate (CP-25) was synthesized to improve the poor oral absorption of paeoniflorin (Pae). 2. This study was performed to investigate the absorptive behavior and mechanism of CP-25 in in situ single-pass intestinal perfusion in rats, using Pae as a control. 3. The results showed that intestinal absorption of CP-25 was neither segmental nor sex dependent. However, the main segment of intestine that absorbed Pae was the duodenum. Furthermore, passive transport was confirmed to be the main absorption pattern of CP-25. More importantly, the absorption of CP-25 was much higher than Pae in the small intestine. 4. Among the ABC transporter inhibitors, the absorption rate of Pae increased in the presence of P-gp inhibitors verapamil and GF120918, which indicated that Pae was a substrate of P-glycoprotein (P-gp), however, such was not observed in the presence of breast cancer resistance protein and multidrug resistance-associated protein 2. Finally, the ABC transporter inhibitors did not have any significant impact on CP-25 as demonstrated in the parallel studies. 5. CP-25 could improve the poor absorption of Pae, which may be attributed to both the lipid solubility enhancement and its resistance to P-gp-mediated efflux.

Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives.

PubMed

Jachez, B; Boesch, D; Grassberger, M A; Loor, F

1993-04-01

FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux. The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype. Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones. FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity. One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

PubMed

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2018-04-01

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Genome-wide identification and expression characterization of ABCC-MRP transporters in hexaploid wheat

PubMed Central

Bhati, Kaushal K.; Sharma, Shivani; Aggarwal, Sipla; Kaur, Mandeep; Shukla, Vishnu; Kaur, Jagdeep; Mantri, Shrikant; Pandey, Ajay K.

2015-01-01

The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots (TaABCC2, TaABCC3, and TaABCC11 and TaABCC12), stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds (TaABCC6, TaABCC8, TaABCC12, TaABCC13, and TaABCC17) implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily. PMID:26191068

Genome-wide identification and expression characterization of ABCC-MRP transporters in hexaploid wheat.

PubMed

Bhati, Kaushal K; Sharma, Shivani; Aggarwal, Sipla; Kaur, Mandeep; Shukla, Vishnu; Kaur, Jagdeep; Mantri, Shrikant; Pandey, Ajay K

2015-01-01

The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots (TaABCC2, TaABCC3, and TaABCC11 and TaABCC12), stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds (TaABCC6, TaABCC8, TaABCC12, TaABCC13, and TaABCC17) implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNAmore » expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.« less

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

PubMed

Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

2008-02-01

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

Efflux-Mediated Drug Resistance in Bacteria: an Update

PubMed Central

Li, Xian-Zhi; Nikaido, Hiroshi

2010-01-01

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome although they can also be plasmid-encoded. A previous article (Li X-Z and Nikaido H, Drugs, 2004; 64[2]: 159–204) had provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past five years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria. PMID:19678712

Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran Sesquiterpenoids as Reversers of P-Glycoprotein-Mediated Multidrug Resistance.

PubMed

Callies, Oliver; Sánchez-Cañete, María P; Gamarro, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Bazzocchi, Isabel L

2016-03-10

P-glycoprotein (P-gp) plays a crucial role in the development of multidrug resistance (MDR), a major obstacle for successful chemotherapy in cancer. Herein, we report on the development of a natural-product-based library of 81 dihydro-β-agarofuran sesquiterpenes (2-82) by optimization of the lead compound 1. The compound library was evaluated for its ability to inhibit P-gp-mediated daunomycin efflux in MDR cells. Selected analogues were further analyzed for their P-gp inhibition constant, intrinsic toxicity, and potency to reverse daunomycin and vinblastine resistances. Analogues 6, 24, 28, 59, and 66 were identified as having higher potency than compound 1 and verapamil, a first-generation P-gp modulator. SAR analysis revealed the size of the aliphatic chains and presence of nitrogen atoms are important structural characteristics to modulate reversal activity. The present study highlights the potential of these analogues as modulators of P-gp mediated MDR in cancer cells.

The structure of the human ABC transporter ABCG2 reveals a novel mechanism for drug extrusion.

PubMed

Khunweeraphong, Narakorn; Stockner, Thomas; Kuchler, Karl

2017-10-23

The human ABC transporter ABCG2 (Breast Cancer Resistance Protein, BCRP) is implicated in anticancer resistance, in detoxification across barriers and linked to gout. Here, we generate a novel atomic model of ABCG2 using the crystal structure of ABCG5/G8. Extensive mutagenesis verifies the structure, disclosing hitherto unrecognized essential residues and domains in the homodimeric ABCG2 transporter. The elbow helix, the first intracellular loop (ICL1) and the nucleotide-binding domain (NBD) constitute pivotal elements of the architecture building the transmission interface that borders a central cavity which acts as a drug trap. The transmission interface is stabilized by salt-bridge interactions between the elbow helix and ICL1, as well as within ICL1, which is essential to control the conformational switch of ABCG2 to the outward-open drug-releasing conformation. Importantly, we propose that ICL1 operates like a molecular spring that holds the NBD dimer close to the membrane, thereby enabling efficient coupling of ATP hydrolysis during the catalytic cycle. These novel mechanistic data open new opportunities to therapeutically target ABCG2 in the context of related diseases.

Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems.

PubMed

Okuda, Ken-ichi; Yanagihara, Sae; Sugayama, Tomomichi; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-06-01

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

PubMed Central

Zelcer, Noam; Saeki, Tohru; Bot, Ilse; Kuil, Annemieke; Borst, Piet

2003-01-01

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis. PMID:12220224

Marine Natural Products with P-Glycoprotein Inhibitor Properties

PubMed Central

Lopez, Dioxelis; Martinez-Luis, Sergio

2014-01-01

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

ABC Transporter Genes and Risk of Type 2 Diabetes

PubMed Central

Schou, Jesper; Tybjærg-Hansen, Anne; Møller, Holger J.; Nordestgaard, Børge G.; Frikke-Schmidt, Ruth

2012-01-01

OBJECTIVE Alterations of pancreatic β-cell cholesterol content may contribute to β-cell dysfunction. Two important determinants of intracellular cholesterol content are the ATP-binding cassette (ABC) transporters A1 (ABCA1) and -G1 (ABCG1). Whether genetic variation in ABCA1 and ABCG1 predicts risk of type 2 diabetes in the general population is unknown. RESEARCH DESIGN AND METHODS We tested whether genetic variation in the promoter and coding regions of ABCA1 and ABCG1 predicted risk of type 2 diabetes in the general population. Twenty-seven variants, identified by previous resequencing of both genes, were genotyped in the Copenhagen City Heart Study (CCHS) (n = 10,185). Two loss-of-function mutations (ABCA1 N1800H and ABCG1 g.-376C>T) (n = 322) and a common variant (ABCG1 g.-530A>G) were further genotyped in the Copenhagen General Population Study (CGPS) (n = 30,415). RESULTS Only one of the variants examined, ABCG1 g.-530A>G, predicted a decreased risk of type 2 diabetes in the CCHS (P for trend = 0.05). Furthermore, when validated in the CGPS or in the CCHS and CGPS combined (n = 40,600), neither the two loss-of-function mutations (ABCA1 N1800H, ABCG1 g.-376C>T) nor ABCG1 g.-530A>G were associated with type 2 diabetes (P values >0.57 and >0.30, respectively). CONCLUSIONS Genetic variations in ABCA1 and ABCG1 were not associated with increased risk of type 2 diabetes in the general population. These data were obtained in general population samples harboring the largest number of heterozygotes for loss-of-function mutations in ABCA1 and ABCG1. PMID:23139370

Characterization and analyses of multidrug resistance-associated protein 1 (MRP1/ABCC1) polymorphisms in Chinese population

PubMed Central

Yin, Ji-Ye; Huang, Qiong; Yang, Youyun; Zhang, Jian-Ting; Zhong, Mei-Zuo; Zhou, Hong-Hao; Liu, Zhao-Qian

2009-01-01

Multidrug resistance (MDR) is one of the major obstacles for successful cancer chemotherapy. Over-expression of ATP-binding cassette (ABC) transporters such as MRP1/ABCC1 has been suggested to cause MDR. In this study, we explored the distribution frequencies of four common single nucleotide polymorphisms (SNPs) of MRP1/ABCC1 in a mainland Chinese population and investigated whether these SNPs affect the expression and function of the MRP1/ABCC1. We found that the allelic frequencies of Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A) and Arg1058Gln (3173G>A) in mainland Chinese were 0.5%, 1.4%, 5.8% and 0.5%, respectively. These four SNPs were recreated by site-directed mutagenesis and tested for their effect on MRP1/ABCC1 expression and MDR function in HEK293 and CHO-K1 cells lines. We found that none of these mutations had any effect on MRP1/ABCC1 expression and trafficking, but that Arg723Gln mutation significantly reduced MRP1/ABCC1-mediated resistance to daunorubicin, doxorubicin, etoposide, vinblastine and vincristine. The Cys43Ser mutation did not affect all tested drugs resistance. On the other hand, the Thr73Ile mutation reduced resistance to methotrexate and etoposide while the Arg1058Gln mutation increased the response of two anthracycline drugs and etoposide in HEK293 and CHO-K1 cells as well as vinblastine and methotrexate in CHO-K1 cells. We conclude that the allelic frequency of the Arg723Gln mutation is relatively higher than other SNPs in mainland Chinese population and therefore this mutation significantly reduces MRP1/ABCC1 activity in MDR. PMID:19214144

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

PubMed

Sletvold, H; Johnsen, P J; Hamre, I; Simonsen, G S; Sundsfjord, A; Nielsen, K M

2008-07-01

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Assessment of vandetanib as an inhibitor of various human renal transporters: inhibition of multidrug and toxin extrusion as a possible mechanism leading to decreased cisplatin and creatinine clearance.

PubMed

Shen, Hong; Yang, Zheng; Zhao, Weiping; Zhang, Yueping; Rodrigues, A David

2013-12-01

Vandetanib was evaluated as an inhibitor of human organic anion transporter 1 (OAT1), OAT3, organic cation transporter 2 (OCT2), and multidrug and toxin extrusion (MATE1 and MATE2K) transfected (individually) into human embryonic kidney 293 cells (HEK293). Although no inhibition of OAT1 and OAT3 was observed, inhibition of OCT2-mediated uptake of 1-methyl-4-phenylpyridinium (MPP(+)) and metformin was evident (IC(50) of 73.4 ± 14.8 and 8.8 ± 1.9 µM, respectively). However, vandetanib was an even more potent inhibitor of MATE1- and MATE2K-mediated uptake of MPP(+) (IC(50) of 1.23 ± 0.05 and 1.26 ± 0.06 µM, respectively) and metformin (IC(50) of 0.16 ± 0.05 and 0.30 ± 0.09 µM, respectively). Subsequent cytotoxicity studies demonstrated that transport inhibition by vandetanib (2.5 µM) significantly decreased the sensitivity [right shift in concentration of cisplatin giving rise to 50% cell death; IC(50(CN))] of MATE1-HEK and MATE2K-HEK cells to cisplatin [IC(50(CN)) of 1.12 ± 0.13 versus 2.39 ± 0.44 µM; 0.85 ± 0.09 versus 1.99 ± 0.16 µM; P < 0.05), but not OCT2-HEK cells (1.36 ± 0.19 versus 1.47 ± 0.24 µM) versus vandetanib untreated cells and Mock-HEK cells [IC(50(CN)) of 2.34 ± 0.31 µM]. In summary, the results show that vandetanib is a potent inhibitor of MATE1 and MATE2K (versus OCT2). Inhibition of the two transporters may explain why there are reports of decreased creatinine clearance, and increased cisplatin nephrotoxicity (reduced cisplatin clearance), in some subjects receiving vandetanib therapy.

The systems biology of uric acid transporters: the role of remote sensing and signaling.

PubMed

Nigam, Sanjay K; Bhatnagar, Vibha

2018-07-01

Uric acid homeostasis in the body is mediated by a number of SLC and ABC transporters in the kidney and intestine, including several multispecific 'drug' transporters (e.g., OAT1, OAT3, and ABCG2). Optimization of uric acid levels can be viewed as a 'systems biology' problem. Here, we consider uric acid transporters from a systems physiology perspective using the framework of the 'Remote Sensing and Signaling Hypothesis.' This hypothesis explains how SLC and ABC 'drug' and other transporters mediate interorgan and interorganismal communication (e.g., gut microbiome and host) via small molecules (e.g., metabolites, antioxidants signaling molecules) through transporters expressed in tissues lining body fluid compartments (e.g., blood, urine, cerebrospinal fluid). The list of uric acid transporters includes: SLC2A9, ABCG2, URAT1 (SLC22A12), OAT1 (SLC22A6), OAT3 (SLC22A8), OAT4 (SLC22A11), OAT10 (SLC22A13), NPT1 (SLC17A1), NPT4 (SLC17A3), MRP2 (ABCC2), MRP4 (ABCC4). Normally, SLC2A9, - along with URAT1, OAT1 and OAT3, - appear to be the main transporters regulating renal urate handling, while ABCG2 appears to regulate intestinal transport. In chronic kidney disease (CKD), intestinal ABCG2 becomes much more important, suggesting remote organ communication between the injured kidney and the intestine. The remote sensing and signaling hypothesis provides a useful systems-level framework for understanding the complex interplay of uric acid transporters expressed in different tissues involved in optimizing uric acid levels under normal and diseased (e.g., CKD, gut microflora dysbiosis) conditions.

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

PubMed Central

Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

2015-01-01

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

PubMed

Li, Rui; Bi, Yi-An; Vildhede, Anna; Scialis, Renato J; Mathialagan, Sumathy; Yang, Xin; Marroquin, Lisa D; Lin, Jian; Varma, Manthena V S

2017-07-01

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC 50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Carrier-mediated transport of riboflavin in the rat colon.

PubMed

Yuasa, H; Hirobe, M; Tomei, S; Watanabe, J

2000-03-01

Carriers involved in riboflavin transport have generally been presumed to be localized in the upper small intestine. However, using a closed loop technique, we found that in the rat colon the absorption of riboflavin could be significantly reduced by raising the concentration from 0.1 to 200 microM and by adding lumiflavin, an analogue of riboflavin. These results suggest that saturable transport by the carrier that is specific for riboflavin and analogues may also be involved in riboflavin absorption in the colon. At the lower concentration of 0.1 microM, carrier-mediated transport was suggested to prevail, compared with passive transport, both in the colon and the small intestine. Furthermore, carrier-mediated transport in the colon was comparable with that in the small intestine. This study is the first to suggest carrier-mediated riboflavin transport in the colon. Although the riboflavin transport system in the colon needs to be subjected to more detailed investigation of its transport functions and role in riboflavin absorption after oral administration, it would be of interest to explore potential use of this carrier as a system for drug delivery.

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

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

PubMed Central

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?

PubMed

Callaghan, Richard; Luk, Frederick; Bebawy, Mary

2014-04-01

P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to "block" P-gp-mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application.

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

PubMed

Weber, J M; Sircar, S; Horvath, J; Dion, P

1989-11-01

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

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

PubMed

Eckford, Paul D W; Sharom, Frances J

2005-07-15

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.

Design, synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors.

PubMed

Wu, Yuxiang; Pan, Miaobo; Dai, Yuxuan; Liu, Baomin; Cui, Jian; Shi, Wei; Qiu, Qianqian; Huang, Wenlong; Qian, Hai

2016-05-15

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC50>100μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

PubMed

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential

Nanomedicine to Deal With Cancer Cell Biology in Multi-Drug Resistance.

PubMed

Tekchandani, Pawan; Kurmi, Balak Das; Paliwal, Shivani Rai

2017-01-01

Today Cancer still remains a major cause of mortality and death worldwide, in humans. Chemotherapy, a key treatment strategy in cancer, has significant hurdles such as the occurrence of chemoresistance in cancer, which is inherent unresponsiveness or acquired upon exposure to chemotherapeutics. The resistance of cancer cells to an antineoplastic agent accompanied to other chemotherapeutic drugs with different structures and mechanisms of action called multi-drug resistance (MDR) plays an important role in the failure of chemo- therapeutics. MDR is primarily based on the overexpression of drug efflux pumps in the cellular membrane, which belongs to the ATP-binding cassette (ABC) superfamily of proteins, are P-gp (P-glycoprotein) and multidrug resistance-associated protein (MRP). Over the years, various strategies have been evaluated to overcome MDR, based not only on the use of MDR modulators but also on the implementation an innovative approach and advanced nanosized drug delivery systems. Nanomedicine is an emerging tool of chemotherapy that focuses on alternative drug delivery for improvement of the treatment efficacy and reducing side effects to normal tissues. This review aims to focus on the details biology, reversal strategies option with the limitation of MDR and various advantages of the present medical science nanotechnology with intracellular delivery aspects for overcoming the significant potential for improving the treatment of MDR malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Research Progress in Reversal of Tumor Multi-drug Resistance via Natural Products.

PubMed

Guo, Qi; Cao, Hongyan; Qi, Xianghui; Li, Huikai; Ye, Peizhi; Wang, Zhiguo; Wang, Danqiao; Sun, Mingyu

2017-11-24

Multidrug resistance occurs when a tumor develops resistance to multiple chemotherapeutic drugs, which may include antitumor drugs with different chemical structures and mechanisms. Multidrug resistance limits the treatment effects of antitumor drugs, and is the main cause of chemotherapy failure. Multidrug resistance is caused by numerous factors including changes in ATP-binding cassette transporters, target proteins, detoxification, deoxyribonucleic acid repair, drug metabolic enzymes, and signal pathways of apoptosis. Clinical research indicates that natural products have great potential to treat tumors and reverse multidrug resistance. Natural products, which often have multiple targets, could play an important role in tumor treatment, have beneficial effects on tumor inhibition, improve symptoms, reduce radiotherapy and chemotherapy side effects, enhance immunity, and prolong survival. Because natural products often have few adverse reactions and less drug resistance, the antitumor activities of natural products have attracted extensive research. We aimed to review the basic research and clinical application of natural products in the reversal of multidrug resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

PubMed

Varmanen, P; Rantanen, T; Palva, A

1996-12-01

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

Suppression of ABCG2 mediated MDR in vitro and in vivo by a novel inhibitor of ABCG2 drug transport.

PubMed

Patel, Atish; Li, Tian-Wen; Anreddy, Nagaraju; Wang, De-Shen; Sodani, Kamlesh; Gadhia, Sanket; Kathawala, Rishil; Yang, Dong-Hua; Cheng, Changmei; Chen, Zhe-Sheng

2017-07-01

Cancer is a disease whose treatment is often limited due to the development of a phenomenon known as multidrug resistance (MDR). There is an immense demand for development of novel agents that can overcome the MDR in cancer. A group of transmembrane proteins called ATP-binding cassette transporters, present ubiquitously in the human body possesses a modular architecture, contributing immensely towards the development of MDR. An analysis of structural congeners among a group of compounds led to the discovery of CCTA-1523 that could selectively reverse ABCG2-mediated MDR in cancer cells in vitro and in vivo. CCTA-1523 (5μM) sensitized the ABCG2 overexpressing cancer cells and ABCG2 transfected cells to the substrate chemotherapeutic drugs. The reversal ability of CCTA-1523 was primarily due to the inhibition of the efflux function of ABCG2; also there was no change in the protein expression or the localization of the ABCG2 in the presence of CCTA-1523. The reversal effect of CCTA-1523 was reversible. Importantly, co-administration of CCTA-1523 restored the in vivo antitumor activity of doxorubicin in ABCG2 overexpressing tumor xenografts. Taken together, our findings indicate that CCTA-1523 is a potent, selective and reversible modulator of ABCG2 that may offer therapeutic promise for multidrug- resistant malignancies. Copyright © 2017 Elsevier Ltd. All rights reserved.

ABC's of Construction. Final Report.

ERIC Educational Resources Information Center

Greater Baton Rouge Chamber of Commerce, LA.

The ABC's of Construction project was a demonstration project designed to integrate basic skills training with an industry-developed vocational-craft training program. The program was located at the central training facility of the Pelican Chapter of Associated Builders and Contractors (ABC), an organization made up of nearly 300 member companies…

Statistical Hypothesis Testing in Intraspecific Phylogeography: NCPA versus ABC

PubMed Central

Templeton, Alan R.

2009-01-01

Nested clade phylogeographic analysis (NCPA) and approximate Bayesian computation (ABC) have been used to test phylogeographic hypotheses. Multilocus NCPA tests null hypotheses, whereas ABC discriminates among a finite set of alternatives. The interpretive criteria of NCPA are explicit and allow complex models to be built from simple components. The interpretive criteria of ABC are ad hoc and require the specification of a complete phylogeographic model. The conclusions from ABC are often influenced by implicit assumptions arising from the many parameters needed to specify a complex model. These complex models confound many assumptions so that biological interpretations are difficult. Sampling error is accounted for in NCPA, but ABC ignores important sources of sampling error that creates pseudo-statistical power. NCPA generates the full sampling distribution of its statistics, but ABC only yields local probabilities, which in turn make it impossible to distinguish between a good fitting model, a non-informative model, and an over-determined model. Both NCPA and ABC use approximations, but convergences of the approximations used in NCPA are well defined whereas those in ABC are not. NCPA can analyze a large number of locations, but ABC cannot. Finally, the dimensionality of tested hypothesis is known in NCPA, but not for ABC. As a consequence, the “probabilities” generated by ABC are not true probabilities and are statistically non-interpretable. Accordingly, ABC should not be used for hypothesis testing, but simulation approaches are valuable when used in conjunction with NCPA or other methods that do not rely on highly parameterized models. PMID:19192182