ABCC Proteins Research Articles

I-CBP112 declines overexpression of ATP-binding cassette transporters and sensitized drug-resistant MDA-MB-231 and A549 cell lines to chemotherapy drugs

Despite extensive efforts and ongoing progress in personalized anticancer approaches, chemotherapy remains the first line or the only treatment for some tumors that may develop resistance to chemotherapeutics in time due to inter alia overexpression of ATP-binding cassette transporters. Using clinically-relevant resistant models of triple negative breast cancer (MDA-MB-231; TNBC) as well as non-small cell lung cancer (A549; NSCLC), we tested the efficacy of I-CBP112 – CBP/EP300 bromodomain inhibitor to overcome drug resistance by declining ABC gene transcription. I-CBP112 significantly reduced ABCB1, ABCC1, ABCC2, ABCC3, ABCC5 and ABCG2 in all resistant lines, as well as ABCC10 in TNBC and ABCC4 in paclitaxel-resistant NSCLC, thereby increasing intracellular drug accumulation and cytotoxicity in 2D and 3D cultures. This was phenocopied only by the joint effect of ABC inhibitors such as tariquidar (ABCB1 – P-glycoprotein and ABCG2) and MK-571 (ABCC), whereas single inhibition of ABCB1/ABCG2 or ABCC proteins did not affect drug accumulation, thereby implying the need of simultaneous deficiency in activity of majority of drug pumps for enhanced drug retention. I-CBP112 failed to directly inhibit activity of ABCB1, ABCG2 and ABCC subfamily members at the same time. Importantly, I-CBP112 treated cancer cells polarized human macrophages into proinflammatory phenotypes. Moreover, I-CBP112 remained non-toxic to primary cell lines, nor did it enhance anticancer drug toxicity to blood-immune cells. In silico assay of ADMET properties confirmed the desired pharmacokinetic features of I-CBP112. The results suggest that the CBP/p300 inhibitor is a promising co-adjuvant to chemotherapy in drug-resistant cancer phenotypes, capable of decreasing ABC transporter expression.

Activity of Lysosomal ABCC3, ABCC5 and ABCC10 is Responsible for Lysosomal Sequestration of Doxorubicin and Paclitaxel-Oregongreen488 in Paclitaxel-Resistant Cancer Cell Lines.

Cancer cell multidrug resistance induced by paclitaxel contributes to the high failure rates of chemotherapy and relapse of the disease. Several mechanisms have been described that underlie the observed resistance, including the overexpression of ABCB1 (P-glycoprotein), which represents an ATP-binding cassette (ABC) transmembrane protein, and its functional occurrence in lysosomal membranes is linked to drug accumulation in these organelles. Using clinically-relevant models of paclitaxel-resistant triple-negative breast cancer and non-small cell lung cancer cell lines, we provide evidence for the role of ABCC subfamily members in the lysosomal sequestration of drugs in multidrug resistant phenotypes. Proteins expression level and its cellular localisation was measured using Western Blot and confocal microscopy. Drug accumulation was analysed by confocal microscopy and flow cytometry. Drug cytotoxicity was tested using resasurin assay and anexin V propidium iodide staining. Regardless of the alteration in gene expression, paclitaxel induced the intracellular redistribution of ABCC3, ABCC5 and ABCC10 and their enrichment in lysosomes. The use of ABCC inhibitors and transient silencing of these three genes limited the accumulation of doxorubicin and paclitaxel-OregonGreen488 in lysosomes, while having little impact on the total drug level inside cells. The cancer cells were also sensitized to various structurally unrelated chemotherapeutics of differing acidity. The results suggest that lysosome membranes anchored ABCC proteins which remained functionally active and were capable to load chemotherapeutics into lysosomes in paclitaxel-resistant cancer cells. Therefore, targeting of lysosomal ABCC transporters may help to overcome paclitaxel-induced resistance by reducing the accumulation of drugs in lysosomes.

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.

Chlorpyrifos stimulates ABCC-mediated transport in the intestine of the rainbow trout Oncorhynchus mykiss

The organophosphorus pesticide chlorpyrifos, detected in water and food worldwide, has also been found in the Río Negro and Neuquén Valley, North Patagonia, Argentina, where the rainbow trout, Oncorhynchus mykiss, is one of the most abundant fish species. We analyzed whether chlorpyrifos affects the transport activity of the ATP-binding cassette protein transporters from the subfamily C (ABCC), which are critical components of multixenobiotic resistance.We exposed ex vivo O. mykiss middle intestine strips (non-polarized) and segments (polarized) for one hour to 0 (solvent control), 3, 10, and 20 μg L−1 and to 0, 10, and 20 μg L−1 chlorpyrifos, respectively. We estimated the Abcc-mediated transport rate by measuring the transport rate of the specific Abcc substrate 2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, we measured the enzymatic activity of cholinesterase, carboxylesterase, glutathione-S-transferase, and 7-ethoxyresorufin-O-deethylase (EROD, indicative of the activity of cytochrome P450 monooxygenase 1A, CYP1A). We also measured lipid peroxidation using the thiobarbituric acid reactive substances method and the gene expression of Abcc2 and genes of the AhR pathway, AhR, ARNT, and cyp1a, by qRT-PCR. Chlorpyrifos induced the DNP-SG transport rate in middle intestine strips in a concentration-dependent manner (49–71%). In polarized preparations, the induction of the DNP-SG transport rate was observed only in everted segments exposed to 20 μg L−1 chlorpyrifos (40%), indicating that CPF only stimulated the apical (luminal) transport flux. Exposure to chlorpyrifos increased GST activity by 42% in intestine strips and inhibited EROD activity (47.5%). In addition, chlorpyrifos exposure inhibited cholinesterase (34–55%) and carboxylesterase (33–42.5%) activities at all the concentrations assayed and increased TBARS levels in a concentration-dependent manner (71–123%). Exposure to 20 μgL−1 chlorpyrifos did not affect the mRNA expression of the studied genes. The lack of inhibition of DNP-SG transport suggests that chlorpyrifos is not an Abcc substrate. Instead, CPF induces the activity of Abcc proteins in the apical membrane of enterocytes, likely through a post-translational pathway.

Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

Genome-wide identification of ABCC gene family and their expression analysis in pigment deposition of fiber in brown cotton (Gossypium hirsutum).

ABC (ATP-binding cassette) transporters are a class of superfamily transmembrane proteins that are commonly observed in natural organisms. The ABCC (ATP-binding cassette C subfamily) protein belongs to a subfamily of the ABC protein family and is a multidrug resistance-associated transporter that localizes to the tonoplast and plays a significant role in pathogenic microbial responses, heavy metal regulation, secondary metabolite transport, and plant growth. Recent studies have shown that the ABCC protein is also involved in the transport of anthocyanins/proanthocyanidins (PAs). To clarify the types and numbers of ABCC genes involved in PA transport in Gossypium hirsutum, the phylogenetic evolution, physical location, and structure of ABCC genes were classified by bioinformatic methods in the upland cotton genome, and the expression levels of these genes were analyzed at different developmental stages of the cotton fiber. The results showed that 42 ABCC genes were initially identified in the whole genome of upland cotton; they were designated GhABCC1-42. The gene structure and phylogenetic analysis showed that the closely related ABCC genes were structurally identical. The analysis of chromosomal localization demonstrated that there were no ABCC genes on the chromosomes of AD/At2, AD/At5, AD/At6, AD/At10, AD/At12, AD/At13, AD/Dt2, AD/Dt6, AD/Dt10, and AD/Dt13. Outside the genes, there were ABCC genes on other chromosomes, and gene clusters appeared on the two chromosomes AD/At11 and AD/Dt8. Phylogenetic tree analysis showed that some ABCC proteins in G. hirsutum were clustered with those of Arabidopsis thaliana, Vitis vinifera and Zea mays, which are known to function in anthocyanin/PA transport. The protein structure prediction indicated that the GhABCC protein structure is similar to the AtABCC protein in A. thaliana, and most of these proteins have a transmembrane domain. At the same time, a quantitative RT-PCR analysis of 42 ABCC genes at different developmental stages of brown cotton fiber showed that the relative expression levels of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 were consistent with the trend of PA accumulation, which may play a role in PA transport. These results provide a theoretical basis for further analysis of the function of the cotton ABCC genes and their role in the transport of PA.

CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to Bacillus thuringiensis Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.)

Rapid evolution of resistance by insect pests severely jeopardizes the sustainable utilization of biopesticides and transgenic crops that produce insecticidal crystal proteins derived from the entomopathogenic bacterium Bacillus thuringiensis (Bt). Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily C genes ABCC2 and ABCC3 in seven lepidopteran insects, including Plutella xylostella (L.). To further determine the causal relationship between alterations in the PxABCC2 and PxABCC3 genes and Cry1Ac resistance in P. xylostella, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct two knockout strains: the ABCC2KO strain is homozygous for a 4-bp deletion in exon 3 of the PxABCC2 gene, and the ABCC3KO strain is homozygous for a 5-bp deletion in exon 3 of the PxABCC3 gene, both of which can produce only truncated ABCC proteins. Bioassay results indicated that high levels of resistance to the Cry1Ac protoxin were observed in both the ABCC2KO (724-fold) and ABCC3KO (413-fold) strains compared to the original susceptible DBM1Ac-S strain. Subsequently, dominance degree and genetic complementation tests demonstrated that Cry1Ac resistance in both the knockout strains was incompletely recessive, and Cry1Ac resistance alleles were located in the classic BtR-1 resistance locus that harbored the PxABCC2 and PxABCC3 genes, similar to the near-isogenic resistant NIL-R strain. Moreover, qualitative toxin binding assays revealed that the binding of the Cry1Ac toxin to midgut brush border membrane vesicles (BBMVs) in both knockout strains was dramatically reduced compared to that in the susceptible DBM1Ac-S strain. In summary, our CRISPR/Cas9-mediated genome editing study presents, for the first time, in vivo reverse genetics evidence for both the ABCC2 and ABCC3 proteins as midgut functional receptors for Bt Cry1 toxins in insects, which provides new insight into the pivotal roles of both the ABCC2 and ABCC3 proteins in the complex molecular mechanism of insect resistance to Bt Cry1 toxins.

Transport of Anthocyanins and other Flavonoids by the Arabidopsis ATP-Binding Cassette Transporter AtABCC2

Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H+-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other’s binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis.

Functional Characterization of ABCC Proteins from Trypanosoma cruzi and Their Involvement with Thiol Transport

Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi and affects 8 million people worldwide. The main chemotherapy is based on benznidazole. The efficacy in the treatment depends on factors such as the parasite strain, which may present different sensitivity to treatment. In this context, the expression of ABC transporters has been related to chemotherapy failure. ABC transporters share a well-conserved ABC domain, responsible for ATP binding and hydrolysis, whose the energy released is coupled to transport of molecules through membranes. The most known ABC transporters are ABCB1 and ABCC1, involved in the multidrug resistance phenotype in cancer, given their participation in cellular detoxification. In T. cruzi, 27 ABC genes were identified in the genome. Nonetheless, only four ABC genes were characterized: ABCA3, involved in vesicular trafficking; ABCG1, overexpressed in strains naturally resistant to benznidazole, and P-glycoprotein 1 and 2, whose participation in drug resistance is controversial. Considering P-glycoprotein genes are related to ABCC subfamily in T. cruzi according to the demonstration using BLASTP alignment, we evaluated both ABCB1-like and ABCC-like activities in epimastigote and trypomastigote forms of the Y strain. The transport activities were evaluated by the efflux of the fluorescent dyes Rhodamine 123 and Carboxyfluorescein in a flow cytometer. Results indicated that there was no ABCB1-like activity in both T. cruzi forms. Conversely, results demonstrated ABCC-like activity in both epimastigote and trypomastigote forms of T. cruzi. This activity was inhibited by ABCC transport modulators (probenecid, indomethacin, and MK-571), by ATP-depleting agents (sodium azide and iodoacetic acid) and by the thiol-depleting agent N-ethylmaleimide. Additionally, the presence of ABCC-like activity was supported by direct inhibition of the thiol-conjugated compound efflux with indomethacin, characteristic of ABCC subfamily members. Taken together, the results provide the first description of native ABCC-like activity in T. cruzi epimastigote and trypomastigote forms, indicating that the study of the biological role for that thiol transporter is crucial to reveal new molecular mechanisms for therapeutic approaches in the Chagas disease.

Abcc10 status affects mammary tumour growth, metastasis, and docetaxel treatment response.

Background:Resistance to chemotherapeutic agents is a major obstacle to cancer treatment. A group of ABC efflux pumps, the Multidrug Resistance Proteins, is a source of resistance. Herein, we investigated the role of ABCC10 in mammary tumours, given the important role we have defined for ABCC10 in transporting taxanes, and the recognition that some ABCC proteins have roles in tumour growth.Methods:ABCC10 expression was correlated to human breast cancer subtype using breast tissue microarrays. Real-time quantitative PCR and western blot analysis were used to examine ABCC10 expression in human breast cancer lines. Abcc10−/− mice were crossed to MMTV-PyVmT mice to produce Abcc10−/− vs Abcc10+/+ mammary tumours and derivative cell lines. We used allograft and cellular assays to perform baseline and drug sensitization analysis of tumours and cell lines.Results:Clinical sample analyses indicated that ABCC10 was more highly expressed in Her2+ and ER+ than in Her2−, ER−, and triple-negative breast cancer. Unexpectedly, PyVmT; Abcc10−/− tumours grew more rapidly than PyVmT; Abcc10+/+ tumours and were associated with significantly reduced apoptosis and metastasis. PyVmT; Abcc10−/− lines were less migratory than PyVmT; Abcc10+/+ lines. Finally, we showed increased survival of docetaxel-treated MMTV-PyVmT; Abcc10−/− mice compared with wild-type mice.Conclusions:These data identify roles for Abcc10 in breast cancer pathogenesis and in vivo docetaxel resistance.

Cellular transport of microcystin-LR in rainbow trout (Oncorhynchus mykiss) across the intestinal wall: Possible involvement of multidrug resistance-associated proteins

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchus mykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs, respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200μM). CDNB enters the cells and is conjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione (DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10min, in order to calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins in microcystin-LR (MCLR) transport, 1.135μM MCLR was added to the bath or inside the sacs, in everted or non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantly inhibited by MCLR. A concentration–response curve obtained using strips from middle intestine yielded an IC50 value of 1.33μM MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibition of DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substrates could bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does not depend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25μM) to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substrate calcein. 2.27μM MCLR and 3μM MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally, MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Middle intestinal segments were incubated in saline solution with 1.135μM MCLR (MC1), 2.27μM MCLR (MC2), 3μM MK571 (MK) or 1.135μM MCLR+3μM MK571 (MC1/MK). After 1h, GSH concentration, protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1did not produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similar proportions (34–49%). MK alone significantly increased PP2A activity (40%) with no effect in any other variable. GST activity and GSH concentration were not affected by any treatment. Concentration–response curves for MCLR (1.135 to 13.62μM) alone or plus 3 or 6μM MK571 were obtained using PP1 activity as response variable. The IC50 values were 1.0, 0.52, and 0.37μM, respectively. Our results suggest that O. mykiss enterocytes are capable of eliminating MCLR by GST-mediated conjugation and luminal excretion through an Abcc-like apical transporter. This mechanism would prevent toxic effects and reduce the toxin uptake into the blood, which is likely mediated by basolateral Abccs.

K ATP Channels and Cardiovascular Disease

ATP-sensitive potassium (KATP) channels were first discovered in the heart 30 years ago. Reconstitution of KATP channel activity by coexpression of members of the pore-forming inward rectifier gene family (Kir6.1, KCNJ8, and Kir6.2 KCNJ11) with sulfonylurea receptors (SUR1, ABCC8, and SUR2, ABCC9) of the ABCC protein subfamily has led to the elucidation of many details of channel gating and pore properties. In addition, the essential roles of Kir6.x and SURx subunits in generating cardiac and vascular KATP(2) and the detrimental consequences of genetic deletions or mutations in mice have been recognized. However, despite this extensive body of knowledge, there has been a paucity of defined roles of KATP subunits in human cardiovascular diseases, although there are reports of association of a single Kir6.1 variant with the J-wave syndrome in the ECG, and 2 isolated studies have reported association of loss of function mutations in SUR2 with atrial fibrillation and heart failure. Two new studies convincingly demonstrate that mutations in the SUR2 gene are associated with Cantu syndrome, a complex multi-organ disorder characterized by hypertrichosis, craniofacial dysmorphology, osteochondrodysplasia, patent ductus arteriosus, cardiomegaly, pericardial effusion, and lymphoedema. This realization of previously unconsidered consequences provides significant insight into the roles of the KATP channel in the cardiovascular system and suggests novel therapeutic possibilities.

Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress

Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages.

Effects of 3β-Acethyl Tormentic Acid (3ATA) on ABCC Proteins Activity

Multidrug resistance (MDR) is considered the main cause of cancer chemotherapy failure and patient relapse. The active drug efflux mediated by transporter proteins of the ABC (ATP-binding cassette) family is the most investigated mechanism leading to MDR. With the aim of inhibiting this transport and circumventing MDR, a great amount of work has been dedicated to identifying pharmacological inhibitors of specific ABC transporters. We recently showed that 3β-acetyl tormentic acid (3ATA) had no effect on P-gp/ABCB1 activity. Herein, we show that 3ATA strongly inhibited the activity of MRP1/ABCC1. In the B16/F10 and Ma104 cell lines, this effect was either 20X higher or similar to that observed with MK571, respectively. Nevertheless, the low inhibitory effect of 3ATA on A549, a cell line that expresses MRP1-5, suggests that it may not inhibit other MRPs. The use of cells transfected with ABCC2, ABCC3 or ABCC4 showed that 3ATA was also able to modulate these transporters, though with an inhibition ratio lower than that observed for MRP1/ABCC1. These data point to 3ATA as a new ABCC inhibitor and call attention to its potential use as a tool to investigate the function of MRP/ABCC proteins or as a co-adjuvant in the treatment of MDR tumors.

Effect of Multiple Cysteine Substitutions on the Functionality of Human Multidrug Resistance Protein 1 Expressed in Human Embryonic Kidney 293 Cells: Identification of Residues Essential for Function

Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

Alternative splicing of an ATP-binding cassette transporter ABCC10 in Tetrahymena thermophila

ATP-binding cassette transporters (ABCT) could generate multiple transcripts through alternative splicing (AS) in mammalian. Some AS introduced PTC (premature terminal codon)-containing isoforms of ABCT couple with NMD (nonsense-mediated mRNA decay) to regulate relevant functions. However, there are no similar reports in lower organisms. This paper focuses on the unicellular protozoa Tetrahymena thermophila, based on the RNA-seq data of Tetrahymena thermophila, identified two alternative splicing variants of gene ABCC10 (SV1 and SV2). The SV2 contained an intron retention event at the fifth intron, and this 49 bp intron resulted in shift-frame and introduced PTC. Then, a knock-down Tetrahymena strain of gene UPF1 which is a key factor of NMD was constructed, and the expression levels of SV2 were performed using a real-time quantitative PCR. The results showed the expression levels of SV2 were up-regulated significantly in knock-down strain, indicating that SV2 was targeted by NMD, which is consistent to the mechanism which the AS introduced PTC-containing isoforms of ABCC proteins can be targeted by NMD in mammalian. Thus, we infer that this mechanism is highly evolutionary conserved in eukaryotes and was already functional in the last eukaryotic common ancestor.

Transmembrane transporters ABCC – structure, function and role in multidrug resistance of cancer cells

Resistance to cytotoxic drugs is a significant problem of systemic treatment of cancers. Apart from drug inactivation, changes in target enzymes and proteins, increased DNA repair and suppression of apoptosis, an important mechanism of resistance is an active drug efflux from cancer cells. Drug efflux across the cell membrane is caused by transport proteins such as ABC proteins (ATP-binding cassette). This review focuses on the ABCC protein subfamily, whose members are responsible for multidrug cross-resistance of cancer cells to cytotoxic agents. The authors discuss the structure of ABCC proteins, their physiological function and diseases provoked by mutations of respective genes, their expression in many different malignancies and its connection with resistance to anticancer drugs, as well as methods of reversion of such resistance.

Knowing Your ABCCs: Novel Functions of ABCC Transporters

Knowing Your ABCCs: Novel Functions of ABCC Transporters

Functional Side Chain Interactions in ABCC Transporters Inferred by Improved Coevolution Analysis

Members of the ABCC sub-family of ABC transport proteins underlie insulin secretion, multidrug resistance in some cancers, and cystic fibrosis disease; unlike most other ABC proteins, they are characterized by two markedly dissimilar ATP binding sites. Recent X-ray structures illuminate numerous inter-residue interactions but it remains largely unknown which interactions are crucial for the signal transduction events that mediate conformational changes during transport. We have inferred such interactions by detecting coevolving pairs of amino-acid positions from aligned ABCC protein sequences using several current methods, which vary in their correction for phylogenetic relatedness, and their assumptions about how amino-acid substitutions occur. We evaluated the performance of these methods by testing their ability to predict side-chain contacts given a 3D structural model. Since the heterogeneity of amino-acid substitution rates generates correlations between positions and thus undermines accurate detection of coevolving pairs, we partitioned pairs into classes based on the rate of amino-acid substitutions at both positions of each pair. We then optimized the performance of the methods in three sequential steps: removal of the most redundant sequences from the alignment to correct for phylogenetic artifacts, logical combination of methods in proportion to their individual performances, and adjustment of the coevolution score of each pair according to the performance associated with that pair's substitution rate class. We verified that performance was improved by these procedures, in comparison to results obtained with each method separately and without partitioning pairs by substitution rate. As expected, many of the detected coevolving pairs bridge consecutive turns in alpha-helices in the structural model. Others, however, link distinct structural elements or domains, and so might transduce signals during the protein's conformational cycle. Intriguingly, some of the positions identified have been implicated in cystic fibrosis.[NIH-DK51767]

An update on the ABCC transporter family in plants: many genes, many proteins, but how many functions?

The ABCC subfamily of the ATP binding cassette (ABC) transporters, which were formerly known as multidrug resistance-related proteins (MRPs), consists of closely related members found in all eukaryotic organisms. Although more than a decade of intensive research has elapsed since the first MRP protein was functionally characterised in Arabidopsis thaliana, knowledge of this particular transporter family is still limited in plants. Although ABCC proteins were originally defined as vacuolar pumps of glutathione-S (GS) conjugates, evidence, as well as speculation, on their endogenous functions inside the cell ranges from detoxification and heavy metal sequestration, to chlorophyll catabolite transport and ion channel regulation. The characterisation of knockout mutants in Arabidopsis has been pivotal for elucidation of different roles of ABCC transporters. However, a functional annotation for the majority of these transport proteins is still lacking, even in this model plant. On the one hand, this problem seems to be caused by functional redundancy between family members, which might lead to physiological complementation by a highly homologous gene in the mutant lines. On the other hand, there is growing evidence that the functional diversity of ABCC genes in Arabidopsis and other plants is far greater than previously assumed. For example, analysis of microarray expression data supports involvement of ABCC transporters in the response to biotic stress: particular changes in ABCC transcript levels are found, which are pathogen-specific and evoke distinct signalling cascades. Current knowledge about plant ABCC transporters indicates that novel and unexpected functions and substrates of these proteins are still waiting to be elucidated.