ATP-binding Cassette Transporter Family Research Articles

Dose-Dependent Physiological and Transcriptomic Responses of Lettuce (Lactuca sativa L.) to Copper Oxide Nanoparticles-Insights into the Phytotoxicity Mechanisms.

Understanding the complex mechanisms involved in plant response to nanoparticles (NPs) is indispensable in assessing the environmental impact of nano-pollutants. Plant leaves can directly intercept or absorb NPs deposited on their surface; however, the toxicity mechanisms of NPs to plant leaves are unclear. In this study, lettuce leaves were exposed to copper oxide nanoparticles (CuO-NPs, 0, 100, and 1000 mg/L) for 15 days, then physiological tests and transcriptomic analyses were conducted to evaluate the negative impacts of CuO-NPs. Both physiological and transcriptomic results demonstrated that CuO-NPs adversely affected plant growth, photosynthesis, and enhanced reactive oxygen species (ROS) accumulation and antioxidant system activity. The comparative transcriptome analysis showed that 2270 and 4264 genes were differentially expressed upon exposure to 100 and 1000 mg/L CuO-NPs. Gene expression analysis suggested the ATP-binding cassette (ABC) transporter family, heavy metal-associated isoprenylated plant proteins (HIPPs), endocytosis, and other metal ion binding proteins or channels play significant roles in CuO-NP accumulation by plant leaves. Furthermore, the variation in antioxidant enzyme transcript levels (POD1, MDAR4, APX2, FSDs), flavonoid content, cell wall structure and components, and hormone (auxin) could be essential in regulating CuO-NPs-induced stress. These findings could help understand the toxicity mechanisms of metal NPs on crops, especially NPs resulting from foliar exposure.

Zebrafish (Danio rerio) ecotoxicological ABCB4, ABCC1 and ABCG2a gene promoters depict spatiotemporal xenobiotic multidrug resistance properties against environmental pollutants

Marine organisms are naturally equipped with multixenobiotic resistance mechanisms that are often governed by ATP-binding cassette (ABC) transporter family members. Previous studies focused on the target genes of ABC but little is known about the functionality of their promoter regions. Due to the importance of promoters in ABC transporter gene regulation, we functionally characterized three major xenobiotic transporter promoters of zebrafish, namely ABCB4, ABCC1 and ABCG2a via in silico transcription factor binding analysis and in vivo spatiotemporal expression analysis. The former revealed the major functional contributors (such as AP-1, C/EBP beta, HNF-1 and NF-1 TFBSs) towards promoter activity enhancement across four different tissues (liver, muscle, cell cycle and immune cells) where majority of them discovered were liver-specific whereas the latter unearthed the localization of these promoters at liver and intestinal tracts during late embryogenesis (48, 72 and 96 hpf). This study contributes towards future xenobiotic transporter ecotoxicology studies in zebrafish.

Identification of novel heavy metal detoxification proteins in Solanum tuberosum: Insights to improve food security protection from metal ion stress.

With increasingly serious environmental pollution problems, research has focused on identifying functional genes within plants that can help ensure food security and soil governance. In particular, plants seem to have been able to evolve specific functional genes to respond to environmental changes by losing partial gene functions, thereby representing a novel adaptation mechanism. Herein, a new category of functional genes was identified and investigated, providing new directions for understanding heavy metal detoxification mechanisms. Interestingly, this category of proteins appears to exhibit specific complexing functions for heavy metals. Further, a new approach was established to evaluate ATP-binding cassette (ABC) transporter family functions using microRNA targeted inhibition. Moreover, mutant and functional genes were identified for future research targets. Expression profiling under five heavy metal stress treatments provided an important framework to further study defense responses of plants to metal exposure. In conclusion, the new insights identified here provide a theoretical basis and reference to better understand the mechanisms of heavy metal tolerance in potato plants. Further, these new data provide additional directions and foundations for mining gene resources for heavy metal tolerance genes to improve safe, green crop production and plant treatment of heavy metal soil pollution.

Overexpression of ABCC1 Confers Drug Resistance to Betulin.

Betulin is a lupane-type pentacyclic triterpene, which is isolated from birch bark. It has a broad spectrum of biological and pharmacological properties, such as anti-inflammatory, anti-tumor, anti-viral, and anti-bacterial activity. Herein, we explored the factors that may result in betulin resistance, especially with respect to its interaction with ATP-binding cassette subfamily C member 1 (ABCC1). ABCC1 is an important member of the ATP-binding cassette (ABC) transporter family, which is central to mediating multidrug resistance (MDR) in naturally derived anticancer agents. An MTT-based cell viability assay showed that ABCC1 overexpression has the ability to desensitize both cancer cell line and gene-transfected cell line to betulin and that this betulin-induced resistance can be antagonized by a known ABCC1 inhibitor MK571 at 25 μM. Additionally, betulin upregulates the ABCC1 protein expression level in both concentration-dependent and time-dependent manners, also blocks the transport function mediated by ABCC1. Subsequently, a high affinity score of betulin was achieved in a computational docking analysis, demonstrating a strong interaction of betulin with ABCC1.

Human drug efflux transporter ABCC5 confers acquired resistance to pemetrexed in breast cancer

AimPemetrexed, a new generation antifolate drug, has been approved for the treatment of locally advanced or metastatic breast cancer. However, factors affecting its efficacy and resistance have not been fully elucidated yet. ATP-binding cassette (ABC) transporters are predictors of prognosis as well as of adverse effects of several xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and can contribute to the optimization of breast cancer treatment regimen.MethodsFirst, we measured the expression levels of ABC transporter family members in cell lines. Subsequently, we assessed the potential role of ABC transporters in conferring resistance to pemetrexed in primary breast cancer cells isolated from 34 breast cancer patients and the role of ABCC5 in mediating pemetrexed transport and apoptotic pathways in MCF-7 cells. Finally, the influence of ABCC5 expression on the therapeutic effect of pemetrexed was evaluated in an in vivo xenograft mouse model of breast cancer.ResultsThe expression levels of ABCC2, ABCC4, ABCC5, and ABCG2 significantly increased in the pan-resistant cell line, and the ABCC5 level in the MCF-7-ADR cell line was 5.21 times higher than that in the control group. ABCC5 expression was inversely correlated with pemetrexed sensitivity (IC50, r = 0.741; p < 0.001) in breast cancer cells derived from 34 patients. Furthermore, we found that the expression level of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cells, with IC50 values of 0.06 and 0.20 μg/mL in ABCC5 knockout and over-expression cells, respectively. In the in vivo study, we observed that ABCC5 affected the sensitivity of pemetrexed in breast tumor-bearing mice, and the tumor volume was much larger in the ABCC5-overexpressing group than in the control group when compared with their own initial volumes (2.7-fold vs. 1.3-fold).ConclusionsOur results indicated that ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and it may serve as a target or biomarker for the optimization of pemetrexed regimen in breast cancer treatment.

Molecular structures of the eukaryotic retinal importer ABCA4.

The ATP-binding cassette (ABC) transporter family contains thousands of members with diverse functions. Movement of the substrate, powered by ATP hydrolysis, can be outward (export) or inward (import). ABCA4 is a eukaryotic importer transporting retinal to the cytosol to enter the visual cycle. It also removes toxic retinoids from the disc lumen. Mutations in ABCA4 cause impaired vision or blindness. Despite decades of clinical, biochemical, and animal model studies, the molecular mechanism of ABCA4 is unknown. Here, we report the structures of human ABCA4 in two conformations. In the absence of ATP, ABCA4 adopts an outward-facing conformation, poised to recruit substrate. The presence of ATP induces large conformational changes that could lead to substrate release. These structures provide a molecular basis to understand many disease-causing mutations and a rational guide for new experiments to uncover how ABCA4 recruits, flips, and releases retinoids.

The functional analysis of ABCG transporters in the adaptation of pigeon pea (Cajanus cajan) to abiotic stresses.

ATP-binding cassette (ABC) transporters are a class of proteins found in living organisms that mediate transmembrane transport by hydrolyzing ATP. They play a vital role in the physiological processes of growth and development in plants. The most numerous sub-type transporter in the ABC transporter family is the ABCG group and which have the most complex function in a plant’s response to abiotic stresses. Our study focused on the effect of ABCG transporters in the adaptation of the pigeon pea to adverse environments (such as drought, salt, temperature, etc.). We conducted a functional analysis of ABCG transporters in the pigeon pea and their role in response to abiotic stresses. A total of 51 ABCG genes (CcABCGs) were identified, and phylogenetic analysis was conducted. We also identified the physicochemical properties of the encoded proteins, predicted their subcellular localization, and identified of the conserved domains. Expression analysis showed that ABCG genes have different expression profiles with tissues and abiotic stresses. Our results showed that CcABCG28 was up-regulated at low temperatures, and CcABCG7 was up-regulated with drought and aluminum stress. The initial results revealed that ABCG transporters are more effective in the abiotic stress resistance of pigeon peas, which improves our understanding of their application in abiotic stress resistance.

Diagnostic and prognostic value of ABC transporter family member ABCG1 gene in clear cell renal cell carcinoma

ABSTRACT As the most common histologic subtype of renal cancer, clear cell renal cell carcinoma (ccRCC) poses a serious threat to public health. However, there are no specific molecular-targeted drugs for ccRCC at present. Human ATP-binding cassette (ABC) transporter family plays an important role in homeostasis maintenance. This study aimed to evaluate the potential diagnostic value of ABC genes in ccRCC. A total of 952 samples of ccRCC patients (707) and controls (245) from three different datasets were included for analysis. Receiver operating characteristic analysis and t-test were used to analyze the differential expression of ABC genes in ccRCC patients and control samples at mRNA level during screening and validations. The Cancer Genome Atlas (TCGA-ccRCC) dataset was utilized to investigate the correlation between ABC genes expression and prognostic value in ccRCC. We then investigated the interactions between ABCG1 and proteins in the Comparative Toxicogenomics Database (CTD). Finally, we found that ATP-binding cassette transporter G member 1 (ABCG1) was over-expressed in ccRCC patients compared with healthy samples at mRNA level. Cox regression analysis and Kaplan–Meier analysis showed that ccRCC patients with high ABCG1 expression had better overall survival (OS) than those patients with low expression (hazard ratio (HR) = 0.662, p = 0.007). This study demonstrated that ABCG1 is a potential diagnostic and prognostic biomarker in ccRCC and discussed the molecular mechanisms underlying the relationship between ccRCC and ABCG1, which might provide guidance for better management and treatment of ccRCC in the future.

Mechanism of Taurohyodeoxycholate-induced Biliary Phospholipid Efflux -Understanding the Function of the ABCB4 Enhancer for Developing Therapeutic Agents against Bile Salt-induced Liver Injury

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Lactate-induced MRP1 expression contributes to metabolism-based etoposide resistance in non-small cell lung cancer cells

BackgroundMetabolic reprogramming contributes significantly to tumor development and is tightly linked to drug resistance. The chemotherapeutic agent etoposide (VP-16) has been used clinically in the treatment of lung cancer but possess different sensitivity and efficacy towards SCLC and NSCLC. Here, we assessed the impact of etoposide on glycolytic metabolism in SCLC and NSCLC cell lines and investigated the role of metabolic rewiring in mediating etoposide resistance.Methodsglycolytic differences of drug-treated cancer cells were determined by extracellular acidification rate (ECAR), glucose consumption, lactate production and western blot. DNA damage was evaluated by the comet assay and western blot. Chemoresistant cancer cells were analyzed by viability, apoptosis and western blot. Chromatin immunoprecipitation (ChIP) was used for analysis of DNA-protein interaction.ResultsHere we showed that exposure to chemotherapeutic drug etoposide induces an exacerbation of ROS production which activates HIF-1α-mediated the metabolic reprogramming toward increased glycolysis and lactate production in non-small cell lung cancer (NSCLC). We identified lactic acidosis as the key that confers multidrug resistance through upregulation of multidrug resistance-associated protein 1 (MRP1, encoded by ABCC1), a member of ATP-binding cassette (ABC) transporter family. Mechanistically, lactic acid coordinates TGF-β1/Snail and TAZ/AP-1 pathway to induce formation of Snail/TAZ/AP-1 complex at the MRP1/ABCC1 promoter. Induction of MRP1 expression inhibits genotoxic and apoptotic effects of chemotherapeutic drugs by increasing drug efflux. Furthermore, titration of lactic acid with NaHCO3 was sufficient to overcome resistance.ConclusionsThe chemotherapeutic drug etoposide induces the shift toward aerobic glycolysis in the NSCLC rather than SCLC cell lines. The increased lactic acid in extracellular environment plays important role in etoposide resistance through upregulation of MRP expression. These data provide first evidence for the increased lactate production, upon drug treatment, contributes to adaptive resistance in NSCLC and reveal potential vulnerabilities of lactate metabolism and/or pathway suitable for therapeutic targeting.2rYXdzaQHsd_zapFCpUg5nVideo Graphical abstractThe chemotherapeutic drug etoposide induces metabolic reprogramming towards glycolysis in the NSCLC cells. The secreted lactic acid coordinates TGF-β1/Snail and TAZ/AP-1 pathway to activate the expression of MRP1/ABCC1 protein, thus contributing to chemoresistance in NSCLC.

Genome-wide identification of ATP binding cassette (ABC) transporter and heavy metal associated (HMA) gene families in flax (Linum usitatissimum L.)

BackgroundThe recent release of the reference genome sequence assembly of flax, a self-pollinated crop with 15 chromosome pairs, into chromosome-scale pseudomolecules enables the characterization of gene families. The ABC transporter and HMA gene families are important in the control of cadmium (Cd) accumulation in crops. To date, the genome-wide analysis of these two gene families has been successfully conducted in some plant species, but no systematic evolutionary analysis is available for the flax genome.ResultsHere we describe the ABC transporter and HMA gene families in flax to provide a comprehensive overview of its evolution and some support towards the functional annotation of its members. The 198 ABC transporter and 12 HMA genes identified in the flax genome were classified into eight ABC transporter and four HMA subfamilies based on their phylogenetic analysis and domains’ composition. Nine of these genes, i.e., LuABCC9, LuABCC10, LuABCG58, LuABCG59, LuABCG71, LuABCG72, LuABCG73, LuHMA3, and LuHMA4, were orthologous with the Cd associated genes in Arabidopsis, rice and maize. Ten motifs were identified from all ABC transporter and HMA genes. Also, several motifs were conserved among genes of similar length, but each subfamily each had their own motif structures. Both the ABC transporter and HMA gene families were highly conserved among subfamilies of flax and with those of Arabidopsis. While four types of gene duplication were observed at different frequencies, whole-genome or segmental duplications were the most frequent with 162 genes, followed by 29 dispersed, 14 tandem and 4 proximal duplications, suggesting that segmental duplications contributed the most to the expansion of both gene families in flax. The rates of non-synonymous to synonymous (Ka/Ks) mutations of paired duplicated genes were for the most part lower than one, indicative of a predominant purifying selection. Only five pairs of genes clearly exhibited positive selection with a Ka/Ks ratio greater than one. Gene ontology analyses suggested that most flax ABC transporter and HMA genes had a role in ATP binding, transport, catalytic activity, ATPase activity, and metal ion binding. The RNA-Seq analysis of eight different organs demonstrated diversified expression profiling patterns of the genes and revealed their functional or sub-functional conservation and neo-functionalization.ConclusionCharacterization of the ABC transporter and HMA gene families will help in the functional analysis of candidate genes in flax and other crop species.

Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors of Human P-glycoprotein.

Human P-glycoprotein (P-gp) is a transmembrane protein that belongs to the ATPBinding Cassette (ABC) transporters family. Physiologically, it exports toxins out of the cell, however, its overexpression leads to the phenomena of Multidrug-Resistance (MDR) by exporting a diverse range of compounds, which are structurally and chemically different from each other, thus creating a hurdle in the treatment of various diseases including cancer. The current study was designed to screen benzophenone sulfonamide derivatives as a class of inhibitors and potential anticancer agents for P-gp. A total number of 15 compounds were evaluated. These compounds were screened in daunorubicin efflux inhibition assays using CCRF-CEM Vcr1000 cell line that overexpressed human P-gp. Cytotoxicity assay was also performed for active compounds 11, 14, and 13. These scaffolds were then docked in the homology model of human P-gp using mouse P-gp as a template (PDB ID: 4MIM) and the recently published Cryo Electron Microscopy (CEM) structure of human mouse chimeric P-gp to find their interactions with specified residues in the binding pocket. Analysis was performed using Labview VI and Graph pad prism version 5.0. Results revealed the potency of all these compounds in low nanomolar range whereas, compound 14 was found to be most active with IC50 value of 18.35nM±4.90 followed by 11 and 13 having IC50 values of 30.66nM±5.49 and 46.12nM±3.06, respectively. Moreover, IC50 values calculated for 14, 11 and 13 in cytotoxicity assay were found to be 22.97μM±0.026, 583.1μM±0.027 and 117.8μM±0.062, respectively. Docking results showed the interaction of these scaffolds in transmembrane helices (TM) where Tyr307, Tyr310, Tyr953, Met986 and Gln946 were found to be the major interaction partners, thus they might play a significant role in the transport of these scaffolds. Benzophenone sulfonamide derivatives showed IC50 values in low nanomolar range comparable to the standard inhibitor Verapamil, therefore they can be good inhibitors of P-gp and can serve as anticancer agents. Also, they have shown interactions in the transmembrane region sharing the same binding region of verapamil and zosuquidar.

Medicinal plants and phytochemicals against multidrug-resistant tumor cells expressing ABCB1, ABCG2, or ABCB5: a synopsis of 2 decades

Multidrug resistance is a major factor causing the failure of cancer chemotherapy. Efflux pumps of the ATP-binding cassette (ABC) transporter family expel a large array of diverse anticancer drugs out of tumor cells thereby rendering them unresponsive to drug treatment. During the past 2 decades, we focused on three ABC transporters, i.e. P-glycoprotein (MDR1/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and ABCB5 with the aim to find natural products with activity against multidrug-resistant cells. We investigated more than 102 cytotoxic medicinal plants and more than 228 isolated cytotoxic phytochemicals with defined chemical structures from 16 countries in Africa, Near East, Asia, and Europe for their activity to kill multidrug-resistant cells. A synopsis of these results is given in this review article. ABCB1-, ABCG2- or ABCB5-expressing cells were moderately or strongly cross-resistant to a considerable portion of plant extracts or phytochemicals, making them less suitable for the treatment of multidrug resistant tumors. While another large portion of compounds inhibited sensitive and multidrug-resistant cells with similar efficacy, a small fraction of medicinal plants and phytochemicals (3–8%) suppressed multidrug-resistant cells with even much better efficacy as their drug-sensitive counterparts. This hypersensitivity phenomenon is termed “collateral sensitivity”. These compounds may be exquisitely suited to kill otherwise resistant and refractory tumors. Molecular modes of action of collateral sensitivity as well as possibilities for further drug development, chemical derivatization of natural lead compounds and rationale phytotherapy are discussed.

Genome sequencing and transcriptome analysis of Geotrichum citri-aurantii on citrus reveal the potential pathogenic- and guazatine-resistance related genes

Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease of citrus，and it causes serious economic losses. In this study, a high-quality genome sequence of G. citri-aurantii was obtained by Single Molecule Real-Time Sequencing (SMRT). Approximately 5.43 Gb of clean data were obtained and a total of 27.94-Mb genomic sequence was mapped to 10 chromosome groups after high-through chromosome conformation capture (Hi-C) assembly. In addition, three polygalacturonase genes which were related to pathogenicity in G. citri-aurantii genome were discovered. And transcriptome data of guazatine-resistance had been analyzed, the results showed that the guazatine-resistance of G. citri-aurantii was related to two ATP-binding cassette (ABC) transporter family genes, six major facilitator superfamily (MFS) transporter family genes and two multidrug and toxic compound extrusion (MATE) transporter family genes. In summary, our research may provide novel insights into the effective control of this pathogen.

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Expression profile of stem cell markers and ABC transporters in 5-fluorouracil resistant Hep-2 cells.

Resistance of laryngeal squamous cell carcinoma cells to traditional therapeutic regimens still remains to be a major reason for therapeutic failure in patients. In this study, we aimed at investigating the expression profiles of ATP-binding cassette (ABC) transporters and stem cell markers in 5-fluorouracil (5-FU) resistant laryngeal Hep-2 cells. We treated parental Hep-2 cells, with stepwise increased doses of 5-FU for almost 1year to develop 5-FU resistant sub-lines with resistance against varying levels of 5-FU concentrations (4 sub-lines resistant to 1, 2, 4, and eightfold of 5-FU). Then, we measured the expression levels of 10 genes from ABC transporters family and 4 stem cell associated markers using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to find out a potential relationship between these markers and chemoresistance. We found that stemness-associated markers had elevated expressions from the beginning of 5-FU resistance acquisition. Their expressions elevated stepwise while parental Hep-2 cells got resistance to higher doses of 5-FU. Expressions of tested ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10 and ABCF2, and ABCG2) were also deregulated in 5-FU resistant Hep-2 cells. Although their expressions remained unaltered at the beginning of acquisition of resistance, expressions of ABC transporters except from ABCB6 increased significantly when cells became resistant to higher doses of 5-FU. Our results suggest that enrichment of cells with stemness characteristics and upregulation of ABC transporters might be amongst the crucial contributors of chemoresistance in laryngeal cancer cells.

A rice chloroplast‐localized ABC transporter ARG1 modulates cobalt and nickel homeostasis and contributes to photosynthetic capacity

Transport and homeostasis of transition metals in chloroplasts, which are accurately regulated to ensure supply and to prevent toxicity induced by these metals, are thus crucial for chloroplast function and photosynthetic performance. However, the mechanisms that maintain the balance of transition metals in chloroplasts remain largely unknown. We have characterized an albino-revertible green 1 (arg1) rice mutant. ARG1 encodes an evolutionarily conserved protein belonging to the ATP-binding cassette (ABC) transporter family. Protoplast transfection and immunogold-labelling assays showed that ARG1 is localized in the envelopes and thylakoid membranes of chloroplasts. Measurements of metal contents, metal transport, physiological and transcriptome changes revealed that ARG1 modulates cobalt (Co) and nickel (Ni) transport and homeostasis in chloroplasts to prevent excessive Co and Ni from competing with essential metal cofactors in chlorophyll and metal-binding proteins acting in photosynthesis. Natural allelic variation in ARG1 between indica and temperate japonica subspecies of rice is coupled with their different capabilities for Co transport and Co content within chloroplasts. This variation underpins the different photosynthetic capabilities in these subspecies. Our findings link the function of the ARG1 transporter to photosynthesis, and potentially facilitate breeding of rice cultivars with improved Co homeostasis and consequently improved photosynthetic performance.

Assessment of AAV Dual Vector Safety in theAbca4-/- Mouse Model of Stargardt Disease.

PurposeAdeno-associated viral (AAV) gene therapy treatment for Stargardt disease currently requires a dual vector approach owing to the size of the ATP-binding cassette transporter family member gene (ABCA4). The nature of the dual vector system creates the potential for adverse events. Here we have investigated an overlapping adeno-associated viral ABCA4 dual vector system for signs of toxicity in Abca4−/− mice as a prelude to dual vector first in human clinical trials.Methods Abca4−/− mice received a subretinal injection of a 1:1 5′:3′ dual vector mix; 5′ vector only; 3′ vector only; a GFP reporter vector; or diluent only (sham). All vectors were adeno-associated virus-8 Y733F. Mice were subsequently assessed for signs of toxicity as measured by loss in retinal structure by optical coherence tomography and retinal function by electroretinography up to 6 months after injection.ResultsSubretinal delivery of the dual vector system and its comprising parts induced no structural or functional changes relative to paired uninjected eyes beyond those observed in the sham control cohort. Histologic changes were limited to the superior retina where the injection was performed. Electroretinography analysis confirmed the dual vector system inferred no functional changes beyond those observed in the sham control cohort.ConclusionsAn optimized overlapping dual vector system for the treatment of Stargardt disease shows no additional signs of toxicity beyond those observed from a sham injection.Translational RelevanceThis presentation of safety of a dual vector system for the treatment of Stargardt disease encourages its future use in clinical trial.

M3814, a DNA-PK Inhibitor, Modulates ABCG2-Mediated Multidrug Resistance in Lung Cancer Cells

M3814, also known as nedisertib, is a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor under phase 2 clinical trials. ABCG2 is a member of the ATP-binding cassette (ABC) transporter family that is closely related to multidrug resistance (MDR) in cancer treatment. In this study, we demonstrated that M3814 can modulate the function of ABCG2 and overcome ABCG2-mediated MDR. Mechanistic studies showed that M3814 can attenuate the efflux activity of ABCG2 transporter, leading to increased ABCG2 substrate drugs accumulation. Furthermore, M3814 can stimulate the ABCG2 ATPase activity in a concentration-dependent manner without affecting the ABCG2 protein expression or cell surface localization of ABCG2. Moreover, the molecular docking analysis indicated a high affinity between M3814 and ABCG2 transporter at the drug-binding cavity. Taken together, our work reveals M3814 as an ABCG2 modulator and provides a potential combination of co-administering M3814 with ABCG2 substrate-drugs to overcome MDR.

Sitravatinib, a Tyrosine Kinase Inhibitor, Inhibits the Transport Function of ABCG2 and Restores Sensitivity to Chemotherapy-Resistant Cancer Cells in vitro.

Sitravatinib, also called MGCD516 or MG-516, is a broad-spectrum tyrosine kinase inhibitor (TKI) under phase III clinical evaluation. Herein, we explored the activity of sitravatinib toward multidrug resistance (MDR) by emphasizing its inhibitory effect on ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is a member of ATP-binding cassette (ABC) transporter family and plays a critical role in mediating MDR. Sitravatinb received an outstanding docking score for binding to the human ABCG2 model (PDB code: 6ETI) among thirty screened TKIs. Also, an MTT assay indicated that sitravatinib at 3 μM had the ability to restore the antineoplastic effect of various ABCG2 substrates in both drug-selected and gene-transfected ABCG2-overexpressing cell lines. In further tritium-labeled mitoxantrone transportation study, sitravatinib at 3 μM blocked the efflux function mediated by ABCG2 and as a result, increased the intracellular concentration of anticancer drugs. Interestingly, sitravatinib at 3 μM altered neither protein expression nor subcellular localization of ABCG2. An ATPase assay demonstrated that ATPase activity of ABCG2 was inhibited in a concentration-dependent manner with sitravatinib; thus, the energy source to pump out compounds was interfered. Collectively, the results of this study open new avenues for sitravatinib working as an ABCG2 inhibitor which restores the antineoplastic activity of anticancer drugs known to be ABCG2 substrates.