Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.
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ABSTRACT:
SUBMITTER: Harris A
PROVIDER: S-EPMC8421411 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.
| S-EPMC6800772 | biostudies-literature
Project description:Substrates of pleiotropic drug resistance (PDR) transporters can induce the expression of corresponding transporter genes by binding to their transcription factors. Penetrating cations are substrates of PDR transporters and theoretically may also activate the expression of transporter genes. However, the accumulation of penetrating cations inside mitochondria may prevent the sensing of these molecules. Thus, whether penetrating cations induce PDR is unclear. Using Saccharomyces cerevisiae as a model, we studied the effects of penetrating cations on the activation of PDR. We found that the lipophilic cation dodecyltriphenylphosphonium (C12TPP) induced the expression of the plasma membrane PDR transporter genes PDR5, SNQ2 and YOR1. Moreover, a 1-hour incubation with C12TPP increased the concentration of Pdr5p and Snq2p and prevented the accumulation of the PDR transporter substrate Nile red. The transcription factor PDR1 was required to mediate these effects, while PDR3 was dispensable. The deletion of the YAP1 or RTG2 genes encoding components of the mitochondria-to-nucleus signalling pathway did not prevent the C12TPP-induced increase in Pdr5-GFP. Taken together, our data suggest (i) that the sequestration of lipophilic cations inside mitochondria does not significantly inhibit sensing by PDR activators and (ii) that the activation mechanisms do not require mitochondria as a signalling module.
| S-EPMC5970188 | biostudies-literature
Project description:Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.
| S-EPMC6349095 | biostudies-literature
Project description:S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resistant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to the transportation of glyphosate in and out of the cell. Taking advantage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells.
| S-EPMC5695762 | biostudies-literature
Project description:Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.
| S-EPMC8727639 | biostudies-literature
Project description: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.
| S-EPMC7908708 | biostudies-literature
Project description:BackgroundIn Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/- cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/- cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/- cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells.Resultsρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain.ConclusionsRPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae.
| S-EPMC8576940 | biostudies-literature
Project description:Doxorubicin is one of the most potent anticancer drugs used in the treatment of various cancer types. The efficacy of doxorubicin is influenced by the drug resistance mechanisms and its cytotoxicity. In this study, we performed a high-copy screening analysis to find genes that play a role in doxorubicin resistance and found several genes (CUE5, AKL1, CAN1, YHR177W and PDR5) that provide resistance. Among these genes, overexpression of PDR5 provided a remarkable resistance, and deletion of it significantly rendered the tolerance level for the drug. Q-PCR analyses suggested that transcriptional regulation of these genes was not dependent on doxorubicin treatment. Additionally, we profiled the global expression pattern of cells in response to doxorubicin treatment and highlighted the genes and pathways that are important in doxorubicin tolerance/toxicity. Our results suggest that many efflux pumps and DNA metabolism genes are upregulated by the drug and required for doxorubicin tolerance.
| S-EPMC4687100 | biostudies-literature
Project description:Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of the PDR5 homolog appeared, linking azole exposure to regulation of this gene.
| S-EPMC105668 | biostudies-literature
Project description:The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
| S-EPMC6911266 | biostudies-literature