Multidrug Resistance ATP-binding Cassette Transporters Research Articles

Heliorhodopsin-mediated light-modulation of ABC transporter

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein–protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Atypical cyclin P regulates cancer cell stemness through activation of the WNT pathway

PurposeCancer stem cells represent a cancer cell subpopulation that has been found to be associated with metastasis and chemoresistance. Therefore, it is vital to identify mechanisms regulating cancer stemness. Previously, we have shown that the atypical cyclin P (CCNP), also known as CNTD2, is upregulated in lung and colorectal cancers and is associated with a worse clinical prognosis. Given that other cyclins have been implicated in pluripotency regulation, we hypothesized that CCNP may also play a role in cancer stemness.MethodsCell line-derived spheroids, ex vivo intestinal organoid cultures and induced-pluripotent stem cells (iPSCs) were used to investigate the role of CCNP in stemness. The effects of CCNP on cancer cell stemness and the expression of pluripotency markers and ATP-binding cassette (ABC) transporters were evaluated using Western blotting and RT-qPCR assays. Cell viability was assessed using a MTT assay. The effects of CCNP on WNT targets were monitored by RNA-seq analysis. Data from publicly available web-based resources were also analyzed.ResultsWe found that CCNP increases spheroid formation in breast, lung and colorectal cancers, and upregulates the expression of stemness (CD44, CD133) and pluripotency (SOX2, OCT4, NANOG) markers. In addition, we found that CCNP promotes resistance to anticancer drugs and induces the expression of multidrug resistance ABC transporters. Our RNA-seq data indicate that CCNP activates the WNT pathway, and that inhibition of this pathway abrogates the increase in spheroid formation promoted by CCNP. Finally, we found that CCNP knockout decreases OCT4 expression in iPSCs, further supporting the notion that CCNP is involved in stemness regulation.ConclusionOur results reveal CCNP as a novel player in stemness and as a potential therapeutic target in cancer.

Ca2+ -activated K+ channel KCa 1.1 as a therapeutic target to overcome chemoresistance in three-dimensional sarcoma spheroid models.

The large‐conductance Ca2+‐activated K+ channel KCa1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three‐dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa1.1 activator‐induced hyperpolarizing responses were significantly larger in human osteosarcoma MG‐63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa1.1 and its role in chemoresistance using a 3D spheroid model. KCa1.1 protein expression levels were significantly elevated in the lipid‐raft‐enriched compartments of MG‐63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa1.1 protein degradation in breast cancer. The siRNA‐mediated inhibition of FBXW7 in MG‐63 cells from 2D monolayers upregulated KCa1.1 protein expression. Furthermore, a treatment with a potent and selective KCa1.1 inhibitor overcame the chemoresistance of the MG‐63 and human chondrosarcoma SW‐1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP‐binding cassette transporters, the expression of the multidrug resistance‐associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa1.1. Therefore, the pharmacological inhibition of KCa1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa1.1‐positive sarcomas.

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.

Allosteric transitions of ATP-binding cassette transporter MsbA studied by the adaptive anisotropic network model.

The transporter MsbA is a kind of multidrug resistance ATP-binding cassette transporter that can transport lipid A, lipopolysaccharides, and some amphipathic drugs from the cytoplasmic to the periplasmic side of the inner membrane. In this work, we explored the allosteric pathway of MsbA from the inward- to outward-facing states during the substrate transport process with the adaptive anisotropic network model. The results suggest that the allosteric transitions proceed in a coupled way. The large-scale closing motions of the nucleotide-binding domains occur first, accompanied with a twisting motion at the same time, which becomes more obvious in middle and later stages, especially for the later. This twisting motion plays an important role for the rearrangement of transmembrane helices and the opening of transmembrane domains on the periplasmic side that mainly take place in middle and later stages respectively. The topological structure plays an important role in the motion correlations above. The conformational changes of nucleotide-binding domains are propagated to the transmembrane domains via the intracellular helices IH1 and IH2. Additionally, the movement of the transmembrane domains proceeds in a nonrigid body, and the two monomers move in a symmetrical way, which is consistent with the symmetrical structure of MsbA. These results are helpful for understanding the transport mechanism of the ATP-binding cassette exporters.

Longitudinal time-dependent effects of irradiation on multidrug resistance in a non-small lung cancer cell line.

Multidrug resistance (MDR) in cancer is known to decrease the therapeutic efficacy of chemotherapy. The effects of irradiation on MDR in cancer cells remain unclear. Tc-99m methoxyisobutylisonitrile (MIBI) exhibits the same ATP-binding cassette (ABC) transporter kinetics as the chemotherapeutic compound doxorubicin. In this study, we investigated the synergistic effects of chemotherapeutics and irradiation [0 Gy: C (control) group; 3, 6, 9, 12 Gy: I (irradiation) group] in the human non-small lung cancer cell line H1299 exhibiting MDR, on MIBI and doxorubicin ABC transporter kinetics, in vitro and in vivo, respectively. In vitro, inhibition of H1299 cell proliferation by irradiation was found to be irradiation dose dependent. The degree and duration of MDR inhibition in vitro in H1299 were also dose dependent. In the cells of both the C group and 3-Gy I group, no significant difference of MIBI accumulation was observed. In the 6-Gy I group, a higher MIBI accumulation was observed at only 7 days after irradiation relative to the C group. A higher MIBI accumulation in the 9- and 12-Gy I groups with a significant difference from the C group was observed at 4 to 14 days after irradiation. A significant negative correlation between intracellular MIBI accumulation and cell replication was found. In vivo, high accumulation and retention of doxorubicin were observed in irradiated tumors in the H1299 xenograft mice group at 4 to 14 days after 9-Gy irradiation compared with the control mice group. These results provide evidence for a synergistic effect of concurrent chemotherapy and radiotherapy.

Functional analysis of ATP-binding cassette transporter of Streptomyces coelicolor

Multidrug resistance (MDR) - type of transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistance to a range of drugs. ATP - binding cassette (ABC) transporters are characterized as MDR transporters utilizing ATP hydrolysis. The SCO 5113 (GeneID: 1100554) encoding a multidrug resistance ABC transporter in Streptomyces coelicolor was expressed in E. coli BL21 (DE3) plysS, using pET 21 a (+). The expression of SCO 5113 occured under 0.1 M IPTG within 2 to 8 hours, was detected on SDS-PAGE at 65 kDa. The E. coli BL21 (DE3) plysS harboring SCO 5113 was used to test for the antibiotic resistance, using disc diffussion method. The results showed the resistance of E. coli BL21 (DE3) plysS harboring SCO 5113 to five kinds of tested antibiotics such as cephalothin (1mg/ml), kanamycin (1mg/ml), ampicillin (10mg/ml), erythromycin (10mg/ml), and chloramphenicol (1mg/ml). The study reported the function of the SCO 5113 gene of Streptomyces coelicolor A3(2). Keywords: ABC transporter, antibiotic resistance, functional analysis, Streptomyces coelicolor Authors: TU NGUYEN, HUU NGUYEN

In vitro investigations of ABC transporters of the human liver – advantages and surprises

The hepatocyte contains a battery of membrane transporters in the sinusoidal and canalicular membrane that guarantee the regulated import and export of nutrients, messengers and toxic compounds. In the canalicular membrane, a number of important ABC (ATP binding cassette) transporters are located. Three of these primary active transporters, BSEP (bile salt export pump, ABC B11), MDR3 (multidrug resistance protein 3, ABC B4) and ABC G5/G8 catalyze the secretion of bile acids (BSEP), phosphatidylcholine (MDR3) and cholesterol (ABC G5/G8). These three molecules form mixed micelles in bile and thereby reduce the harmful action of unbound bile salts on membranes. BSEP, which was initially named s-Pgp (sister of P-gp) and MDR3 share a high sequence identity with MDR1 (P-glycoprotein, ABC B1), which is the hallmark of the family of multidrug resistance ABC transporter present in every eukaryotic cell. For BSEP the identity is approximately 70% and for MDR3 even 85%, respectively. Nevertheless, MDR3 is specific for phosphatidylcholine lipids and BSEP is specific for bile acids, while MDR1 is capable of translocating a myriad of structurally unrelated compound across biological membranes. This of course raises the question, which residues impose specificity in the case of BSEP and MDR3 and which residues impose promiscuity in the case of MDR1. Homology models of BSEP and MDR3 were generated based on the crystal structures of Sav1866 and mouse P-gp to obtain initial information about the putative three-dimensional location of certain amino acid residues of BSEP and MDR3. However, one has to stress that these “structures” represent only models that have to be used with proper caution. The homology model of MDR3 was helpful in providing a structural explanation for the non-functionality of the MDR3 mutant H1231Y. The mutant protein was properly located to the plasma membrane, but displayed a severe substrate transport phenotype. Histidine 1231 corresponds to the histidine of the H-loop, a residue that is critical for ATP hydrolysis in many other ABC transporter systems. Thus, exchange of His against Tyr in position 1231 of MDR3 will impair ATP hydrolysis in one of the two ATP binding site drastically reducing transport activity of MDR3 H1231Y [1]. To study the transport cycle and the importance of mutations within these transporters on their function, overexpression systems are required. We decided to use the methylotrophic yeast Pichia pastoris for the overexpression of two of the three transporters, BSEP and MDR3, respectively. Initially we encountered severe problems in cloning the transporters genes in E. coli employing standard approaches. Only after establishing a cloning strategy that is entirely based on Saccharomyces cerevisiae, both genes could be successfully cloned [2]. This allowed us to combine the speed and ease of cloning of this yeast system for the wild type genes and mutations that were identified in patients. Having this tool in hand, we overexpressed BSEP in yeast to study its transport function in plasma membrane vesicles. This allowed us to establish structure-function relations for a new clinically relevant mutation (G374S) that resulted in a phenotype between PFIC-2 and BRIC-2. Although membrane targeting was normal, e.g. targeting to the canalicular membrane was evident, transport activity of the G374S was strongly impaired [3]. The Sav1866-based homology model of BSEP suggested that Gly 374 is lining the translocation pore of BSEP. Thus, an exchange to serine would impose steric restriction within the pore, thereby influencing the substrate spectrum as evident from the bile acid transport assays. Based on the expression levels of BSEP and MDR3, purification protocols for both membrane proteins were successfully established [4]. Thus, next to the transport assay that is based only highly enriched yeast plasma membranes, purified protein is now also available in the detergent-solubilized state. While the transport assay can be used for BSEP to analyze a possible influence of a mutation on the transporter activity of the protein, purified MDR3 can be used to determine the influence of a mutation on ATPase activity and / or stimulation or inhibition of this activity. In summary, the established yeast overexpression system combines the ease and speed of yeast cloning approaches with the possibility to overexpress and purify both proteins in a quantity and homogeneity sufficient for biochemical and biophysical investigations of the function of these two ABC transporters. Furthermore, the speed with which mutants can be obtained will allow an in depth characterization of clinically relevant mutations of both transporters with respect to function and spectrum of transported substrates.

Synthesis of 5-oxyquinoline derivatives for reversal of multidrug resistance

The inhibition of ABC (ATP binding cassette) transporters is considered a powerful tool to reverse multidrug resistance. Zosuquidar featuring a difluorocyclopropyl-annulated dibenzosuberyl moiety has been found to be an inhibitor of the P-glycoprotein, one of the best-studied multidrug efflux pumps. Twelve 5-oxyisoquinoline derivatives, which are analogues of zosuquidar wherein the dibenzosuberyl-piperazine moiety is replaced by either a diarylaminopiperidine or a piperidone-derived acetal or thioacetal group, have been synthesized as pure enantiomers. Their inhibitory power has been evaluated for the bacterial multidrug-resistance ABC transporter LmrCD and fungal Pdr5. Four of the newly synthesized compounds reduced the transport activity to a higher degree than zosuquidar, being up to fourfold more efficient than the lead compound in the case of LmrCD and about two times better for Pdr5.

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

BackgroundThe 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.ResultsA 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.ConclusionsPDR18 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 fermentation performance for sustainable bio-ethanol production.

Lipid raft involved in drug resistance：relationship between multidrug resistance ATP-binding cassette transporters and lipid raft

Lipid rafts have been implicated in many cellular functions,including protein and lipid transport and signal transduction.Recently ATP-binding cassette(ABC) transporters,which are associated with multidrug resistance,have been found in lipid rafts;therefore they might be related to drug resistance.Here we introduce the relationship between the localization and functions of three multi-drug related ABC transporters,including two relevant to multidrug resistance in tumor cells(Pgp/ABCB1 and MRP1/ABCC1) and one relevant to multidrug resistance in Candida albicans(Cdr1p).We also discuss the influence of sphingolipids and cholesterol,two major components of lipid rafts,on the localization and function of the above three ABC transporters.

Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult-to-treat infections and exhibits significant degrees of poorly understood multidrug resistance (MDR). The aim of this study was to identify and characterize a multidrug ATP-binding cassette (ABC) efflux pump in S. maltophilia. SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine. Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E. coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors. SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.

Inhibitors of cancer cell multidrug resistance mediated by breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP-binding cassette (ABC) transporter superfamily. It is able to efflux a broad range of anti-cancer drugs through the cellular membrane, thus limiting their anti-proliferative effects. Due to its relatively recent discovery in 1998, and in contrast to the other ABC transporters P-glycoprotein (MDR1/ABCB1) and multidrug resistance-associated protein (MRP1/ABCC1), only a few BCRP inhibitors have been reported. This review summarizes the known classes of inhibitors that are either specific for BCRP or also inhibit the other multidrug resistance ABC transporters. Information is presented on structure-activity relationship aspects and how modulators may interact with BCRP.

Increased Expression of Two Multidrug Transporter-Like Genes Is Associated with Ethidium Bromide and Ciprofloxacin Resistance in Mycoplasma hominis

Two genes, md1 and md2, coding for multidrug resistance ATP-binding cassette transporters were identified in Mycoplasma hominis PG21. Expression of these two genes, quantified by quantitative competitive reverse transcription-PCR, was significantly increased in ethidium bromide-resistant strains of M. hominis compared to that in M. hominis PG21.

Overexpression of LaMDR2, a novel multidrug resistance ATP-binding cassette transporter, causes 5-fluorouracil resistance in Leishmania amazonensis

The ATP-binding cassette (ABC) proteins play an important role in drug resistance and detoxification in various organisms. Here we isolated LaMDR2, a new member of the multidrug resistance (MDR) subfamily of ABC proteins in Leishmania amazonensis. LaMDR2 exhibited 47% amino acid identity to its most closely related protein, LaMDR1, which was previously isolated from the same species. Promastigotes that overexpressed LaMDR2 showed significant resistance to 5-fluorouracil (5-FU), but not to LaMDR1 substrates. Expression of LaMDR2 in the transfectants was relatively higher in the log phase than the stationary phase, and a lower accumulation of [ 3H]5-FU was observed in the log-phase cells. These results suggest that LaMDR2 is involved in extrusion of xenobiotics, but functionally different from LaMDR1.

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.