scholarly journals Regulation of the pleiotropic drug resistance transcription factors Pdr1 and Pdr3 in yeast

St open ◽  
2021 ◽  
Vol 2 ◽  
pp. 1-17
Author(s):  
Tea Vasiljević ◽  
Markus Proft
Keyword(s):  
Drug Resistance ◽  
Chromatin Immunoprecipitation ◽  
Cell Model ◽  
Degradation Mechanism ◽  
Model Organism ◽  
Protein Detection ◽  
Eukaryotic Cell ◽  
Pleiotropic Drug Resistance ◽  
Treat Ment ◽  
Different Strains

Aim: To understand how transcriptional factors Pdr1 and Pdr3, belonging to the pleiotropic drug resistance system, are activated, and regulated after introducing chemical tox- ins to the cell in the model organism Saccharomyces cere-visiae. Methods: Series of molecular methods were applied using different strains ofS. cerevisiae over-expressing proteins of interest as a eukaryotic cell model. The chemical stress in- troduced to the cell is represented by menadione. Results were obtained performing protein detection and analysis. Additionally, the regulation of the DNA binding of the tran- scriptional activators after stimulation is quantified using chromatin immunoprecipitation, employing epitope-tagged factors and real-time qPCR. Results: Our results indicated higher expression levels of the Pdr1 transcriptional factor, compared to its homolo- gous Pdr3 after treatment with menadione. The yeast-cell defence system was tested against various organic solvents to exclude the possibility of their presence potentially af- fecting the results. The results indicate that Pdr1 is most abundant after 30 minutes from the beginning of the treat- ment, compared with 240 minutes after the treatment when the function of the transcription factor is faded. It appears that Pdr1 binding to the PDR5 and SNQ2 promoters, which are both activated by Pdr1, peaks around the same time, or more precisely after 40 minutes from the start of the treatment. Conclusion: The tendency of Pdr1 reduction after its activa- tion by menadione is detected. One possibility is that Pdr1, after recognizing the xenobiotic menadione, is removed by a degradation mechanism. Given the fact that Pdr1 directly binds the xenobiotic molecule, its destruction might help the cells to remove toxic levels of menadione. It is possible that overexpressing the part of Pdr1 which recognizes me- nadione alone was sufficient to detoxify and hence produce a tolerance towards menadione.

scholarly journals Penetrating cations induce pleiotropic drug resistance in yeast

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Kseniia V. Galkina ◽  
Elizaveta G. Besedina ◽  
Roman A. Zinovkin ◽  
Fedor F. Severin ◽  
Dmitry A. Knorre
Keyword(s):  
Drug Resistance ◽  
Pleiotropic Drug Resistance ◽  
Penetrating Cations

Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms

2015 ◽  
Vol 11 (11) ◽  
pp. 3129-3136 ◽  
Author(s):  
Namal V. C. Coorey ◽  
James H. Matthews ◽  
David S. Bellows ◽  
Paul H. Atkinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Mechanism Of Action ◽  
Gene Deletion ◽  
Genomic Library ◽  
Deletion Mutants ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA).

scholarly journals The Ribosomal Protein L28 Gene Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Yi Shi ◽  
Xiaojiang Wang ◽  
Qiong Zhu ◽  
Gang Chen
Keyword(s):  
Hepatocellular Carcinoma ◽  
Drug Resistance ◽  
Hepg2 Cells ◽  
Cell Model ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Resistant Gene ◽  
Key Genes ◽  
Resistant Cells

Abstract Background: Sorafenib is the first molecular-targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its treatment efficiency decreases after a short period of time because of the development of drug resistance. This study investigates the role of key genes in regulating sorafenib-resistance in hepatocellular carcinoma and elucidates the mechanism of drug resistance. Methods: The HCC HepG2 cells were used to generate a sorafenib-resistant cell model by culturing the cells in gradually increasing concentration of sorafenib. RNA microarray was applied to profile gene expression and screen key genes associated with sorafenib resistance. Specific targets were knockdown in sorafenib-resistant HepG2 cells for functional studies. The HCC model was established in ACI rats using Morris hepatoma3924A cells to validate selected genes associated with sorafenib resistance in vivo. Results: The HepG2 sorafenib-resistant cell model was successfully established. The IC50 of sorafenib was 9.988mM in HepG2 sorafenib-resistant cells. A total of 35 up-regulated genes were detected by expression profile chip. High-content screening technology was used and a potential drug-resistant gene RPL28 was filtered out. After knocking down of RPL28 in HepG2 sorafenib-resistant cells, the results of cell proliferation and apoptosis illustrated that RPL28 is the key drug-resistant gene in the cells. Furthermore, it was found that both RNA and protein expression of RPL28 increased in HepG2 sorafenib-resistant specimens of Morris Hepatoma rats. In addition, the expression of functional proteins Ki-67 increased in sorafenib-resistant cells. Conclusion: Our study suggested that RPL28 was a key gene for sorafenib resistance in HCC both in vitro and in vivo.

scholarly journals The Road Less Traveled? Unconventional Protein Secretion at Parasite–Host Interfaces

2021 ◽  
Vol 9 ◽  
Author(s):  
Erina A. Balmer ◽  
Carmen Faso
Keyword(s):  
Protein Secretion ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Model ◽  
Signal Sequence ◽  
Model Organism ◽  
Secreted Proteins ◽  
Current Status ◽  
Secretory Proteins ◽  
Secretion Pathways

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.

scholarly journals Genome-Wide Screening and Genetic Networks in Pleiotropic Drug Resistance, Oxidative Stress, and Drug Targets in S. Cerevisiae

2021 ◽  
Author(s):  
◽  
Ploi Yibmantasiri
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Abc Transporters ◽  
Oxidative Stress Response ◽  
Genetic Networks ◽  
Antifungal Compound ◽  
Pleiotropic Drug Resistance ◽  
Gene Deletions ◽  
Genome Wide ◽  
And Oxidative Stress

<p>One of the major problems in biology is to identify genes that are involved in specific processes. Classical genetics and biochemistry, although powerful and informative, can be very labour intensive and do not necessarily characterise networked genes in processes that may overarch numerous biochemical pathways. Here we utilised genomic tools that are capable of defining networks to identify genes involved the complex target mode-of-action of a novel antifungal compound, neothyonidioside and in regulating specific stress processes and the PDR phenotype. The first part of this study investigated the mode-of-action of the antifungal compound, neothyonidioside (neo). We developed a neo resistant mutant strain then utilising a modification of SGAM, a genetic mapping tool, and application of genome-wide chemical-genetic profiling, we identified the neo resistant locus NCP1. This gene acts at a late step in ergosterol biosynthesis but is not the target of neo. The finding that many of the component genes in the ESCRT complex were necessary for neo resistance allowed us to predict and verify by high-content fluorescence microcopy that interruptions in the endosome-multivesicular body pathway were involved. From the known function of the ESCRT proteins and that neo binds ergosterol only above threshold concentrations of ergosterol (explaining the mutant phenotype) we concluded that neo disruption of membrane curvature and fusion capability in the endosome-vacuole pathway is its target. In the second part of this study we identified genes in a genome-wide fashion that modulate the pleiotropic drug resistance (PDR) phenotype and oxidative stress response. Many PDR targets are well studied ABC transporters (e.g. PDR5 , YOR1), but the modulating events between xenobiotic sensing and transcription factor activation, and possible crosstalk between PDR and other stress responses such as oxidative stress are not well characterised. To identify specific genes involved in the PDR and oxidative stress processes, we developed a fluorescent reporter screen for effects on the PDR-target ABC-transporters, Pdr5p and Yor1p tagged with GFP. For the oxidative stress response, the oxidative stress (OS) transcription factor Yap1p tagged with GFP was used. Each reporter was placed in the yeast non-essential gene deletion background of ~4800 strains which were then subjected to either xenobiotic treatments (PDR –GFP reporters) or oxidant treatments (Yap1p-GFP). We then screened for gene deletions which prevented the normal upregulation of PDR reporters in the presence of xenobiotics. Controls were included in the screens that assured we were assessing genes that must contribute to or act before the transcription of the ABC-transporters. A similar screening strategy was pursued for identifying gene deletions that prevent the normal nuclear re-localisation of Yap1p in the presence of oxidants. A major finding in this study was identification of genes contributing to the PDR phenotype that involved signalling (Rho-GTPase, MAPK), that were involved in RNA polymerase II mediator complexes and chromatin modification (subunits of ADA and SAGA histone acetyltransferase complexes), and that were involved in sphingo/phosphorlipids biosynthesis. Secondary screens comprising spot dilution growth assays and Western blots of Pdr5p abundance confirmed key genes of the primary screen and showed that these were specific and not global transcriptional effects.For some of the gene-dependencies, our results can only be construed to indicate the existence of alternative pathways underpinning the PDR phenotype in a Pdr1p/Pdr3p independent manner. We then supposed that if in fact PDR phenotypes are the result of genetic networks, then genes known to interact with the most highly connected hubs from our PDR screen results should also to some extent contribute to the PDR phenotype (spot dilution growth assays, Western blot abundance). A selection of 18 such genes that also appeared in our primary screen but were deemed to be below the cut-off point were phenotype tested and in 60% of the cases showed similar phenotypes to the genes already identified. This result not only proved the validity of the screening methods but validated the original supposition, i.e. that PDR phenotypes can be affected, through gene networks.</p>

scholarly journals New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

2002 ◽  
Vol 22 (8) ◽  
pp. 2642-2649 ◽  
Author(s):  
Stéphane Le Crom ◽  
Frédéric Devaux ◽  
Philippe Marc ◽  
Xiaoting Zhang ◽  
W. Scott Moye-Rowley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Binding Site ◽  
Time Course ◽  
Target Genes ◽  
Regulation System ◽  
Dependent Manner ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

scholarly journals A Member of the PLEIOTROPIC DRUG RESISTANCE Family of ATP Binding Cassette Transporters Is Required for the Formation of a Functional Cuticle in Arabidopsis

2011 ◽  
Vol 23 (5) ◽  
pp. 1958-1970 ◽  
Author(s):  
Michael Bessire ◽  
Sandra Borel ◽  
Guillaume Fabre ◽  
Luis Carraça ◽  
Nadia Efremova ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Atp Binding ◽  
Pleiotropic Drug Resistance ◽  
Atp Binding Cassette Transporters ◽  
Atp Binding Cassette
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