Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

Phosphate Regulated Proteins Of Xanthomonas Citri Subsp. Citri: A Proteomic Approach.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

Impact of cholesterol on ABC and SLC transporters expression and function and its role in disposition variability to lipid-lowering drugs

This report focuses on the effects of cholesterol on the expression and function of the ATP-binding cassette (ABCB1, ABCG2 and ABCC2) and solute-linked carrier (SLCO1B1 and SLCO2B1) drug transporters with a particular focus on the potential impact of cholesterol on lipid-lowering drug disposition. Statins are the most active agents in the treatment of hypercholesterolemia. However, considerable interindividual variation exists in the response to statin therapy. Therefore, it would be huge progress if factors were identified that reliably differentiate between responders and nonresponders. Many studies have suggested that plasma lipid concentrations can affect drug disposition of compounds, such as ciclosporin and amphotericin B. Both compounds are able to affect the expression and function of ABC transporters. Although still speculative, these effects might be owing to the regulation of drug transporters by plasma cholesterol levels. Studies with normo- and hyper-cholesterolemic individuals, before and after atorvastatin treatment, have demonstrated that plasma cholesterol levels are correlated with drug transporter expression, as well as being related to atorvastatin`s cholesterol-lowering effect. The mechanism influencing the correlation between cholesterol levels and the expression and function of drug transporters remains unclear. Some studies provide strong evidence that nuclear receptors, such as the pregnane X receptor and the constitutive androstane receptor, mediate this effect. In the near future, pharmacogenomic studies with individuals in a pathological state should be performed in order to identify whether high plasma cholesterol levels might be a factor contributing to interindividual oral drug bioavailability.

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Antifungal Resistance Mechanisms in Dermatophytes

Although fungi do not cause outbreaks or pandemics, the incidence of severe systemic fungal infections has increased significantly, mainly because of the explosive growth in the number of patients with compromised immune system. Thus, drug resistance in pathogenic fungi, including dermatophytes, is gaining importance. The molecular aspects involved in the resistance of dermatophytes to marketed antifungals and other cytotoxic drugs, such as modifications of target enzymes, over-expression of genes encoding ATP-binding cassette (ABC) transporters and stress-response-related proteins are reviewed. Emphasis is placed on the mechanisms used by dermatophytes to overcome the inhibitory action of terbinafine and survival in the host environment. The relevance of identifying new molecular targets, of expanding the understanding about the molecular mechanisms of resistance and of using this information to design new drugs or to modify those that have become ineffective is also discussed.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

BACKGROUND AND AIMS Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata.

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes.

The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible C. albicans (O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the CDR1, CDR2, CaMDR1, and FLU1 transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (P = 0.05 at 48 h with an inoculum of 10(3) CFU/ml) and a more rapid fungicidal effect of FLC-CY (P = 0.05 at 24 h with an inoculum of 10(3) CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (P = 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (P = 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a "dual-hit" mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by CDR1, CDR2, CaMDR1, and FLU1 genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Gene expression profile of ABC transporters and cytotoxic effect of ibuprofen and acetaminophen in an epithelial ovarian cancer cell line in vitro

PURPOSES: To determine the basic expression of ABC transporters in an epithelial ovarian cancer cell line, and to investigate whether low concentrations of acetaminophen and ibuprofen inhibited the growth of this cell line in vitro. METHODS: TOV-21 G cells were exposed to different concentrations of acetaminophen (1.5 to 15 μg/mL) and ibuprofen (2.0 to 20 μg/mL) for 24 to 48 hours. The cellular growth was assessed using a cell viability assay. Cellular morphology was determined by fluorescence microscopy. The gene expression profile of ABC transporters was determined by assessing a panel including 42 genes of the ABC transporter superfamily. RESULTS: We observed a significant decrease in TOV-21 G cell growth after exposure to 15 μg/mL of acetaminophen for 24 (p=0.02) and 48 hours (p=0.01), or to 20 μg/mL of ibuprofen for 48 hours (p=0.04). Assessing the morphology of TOV-21 G cells did not reveal evidence of extensive apoptosis. TOV-21 G cells had a reduced expression of the genes ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 within the ABC transporter superfamily. CONCLUSIONS: This study provides in vitro evidence of inhibitory effects of growth in therapeutic concentrations of acetaminophen and ibuprofen on TOV-21 G cells. Additionally, TOV-21 G cells presented a reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 transporters.