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1.
Sci Rep ; 14(1): 11695, 2024 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-38778133

RESUMEN

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.


Asunto(s)
Simulación del Acoplamiento Molecular , Saccharomyces cerevisiae , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Antagonistas del Ácido Fólico/farmacología , ARN/metabolismo , Humanos , Fungicidas Industriales/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Biology (Basel) ; 13(3)2024 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-38534408

RESUMEN

Maintaining translation fidelity is a critical step within the process of gene expression. It requires the involvement of numerous regulatory elements to ensure the synthesis of functional proteins. The efficient termination of protein synthesis can play a crucial role in preserving this fidelity. Here, we report on investigating a protein of unknown function, YNR069C (also known as BSC5), for its activity in the process of translation. We observed a significant increase in the bypass of premature stop codons upon the deletion of YNR069C. Interestingly, the genomic arrangement of this ORF suggests a compatible mode of expression reliant on translational readthrough, incorporating the neighboring open reading frame. We also showed that the deletion of YNR069C results in an increase in the rate of translation. Based on our results, we propose that YNR069C may play a role in translation fidelity, impacting the overall quantity and quality of translation. Our genetic interaction analysis supports our hypothesis, associating the role of YNR069C to the regulation of protein synthesis.

3.
FASEB J ; 38(5): e23439, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38416461

RESUMEN

Reactive oxygen species (ROS) are among the most severe types of cellular stressors with the ability to damage essential cellular biomolecules. Excess levels of ROS are correlated with multiple pathophysiological conditions including neurodegeneration, diabetes, atherosclerosis, and cancer. Failure to regulate the severely imbalanced levels of ROS can ultimately lead to cell death, highlighting the importance of investigating the molecular mechanisms involved in the detoxification procedures that counteract the effects of these compounds in living organisms. One of the most abundant forms of ROS is H2 O2 , mainly produced by the electron transport chain in the mitochondria. Numerous genes have been identified as essential to the process of cellular detoxification. Yeast YAP1, which is homologous to mammalian AP-1 type transcriptional factors, has a key role in oxidative detoxification by upregulating the expression of antioxidant genes in yeast. The current study reveals novel functions for COX5A and NPR3 in H2 O2 -induced stress by demonstrating that their deletions result in a sensitive phenotype. Our follow-up investigations indicate that COX5A and NPR3 regulate the expression of YAP1 through an alternative mode of translation initiation. These novel gene functions expand our understanding of the regulation of gene expression and defense mechanism of yeast against oxidative stress.


Asunto(s)
Aterosclerosis , Proteínas de Saccharomyces cerevisiae , Animales , Saccharomyces cerevisiae/genética , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno , Antioxidantes , Mamíferos , Factores de Transcripción/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-36675300

RESUMEN

Lithium chloride (LiCl) has been widely researched and utilized as a therapeutic option for bipolar disorder (BD). Several pathways, including cell signaling and signal transduction pathways in mammalian cells, are shown to be regulated by LiCl. LiCl can negatively control the expression and activity of PGM2, a phosphoglucomutase that influences sugar metabolism in yeast. In the presence of galactose, when yeast cells are challenged by LiCl, the phosphoglucomutase activity of PGM2p is decreased, causing an increase in the concentration of toxic galactose metabolism intermediates that result in cell sensitivity. Here, we report that the null yeast mutant strains DBP7∆ and YRF1-6∆ exhibit increased LiCl sensitivity on galactose-containing media. Additionally, we demonstrate that DBP7 and YRF1-6 modulate the translational level of PGM2 mRNA, and the observed alteration in translation seems to be associated with the 5'-untranslated region (UTR) of PGM2 mRNA. Furthermore, we observe that DBP7 and YRF1-6 influence, to varying degrees, the translation of other mRNAs that carry different 5'-UTR secondary structures.


Asunto(s)
Cloruro de Litio , Proteínas de Saccharomyces cerevisiae , Cloruro de Litio/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Galactosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ARN Helicasas DEAD-box/metabolismo
5.
Biochem Biophys Res Commun ; 641: 110-115, 2023 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-36527744

RESUMEN

Despite the similarity in fundamental goals of translation initiation between different domains of life, it is one of the most phylogenetically diverse steps of the central dogma of molecular biology. In a classical view, the translation signals for prokaryotes and eukaryotes are distinct from each other. This idea was challenged by the finding that the Internal Ribosome Entry Site (IRES) belonging to Plautia stali intestine virus (PSIV) could bypass the domain-specific boundaries and effectively initiate translation in E. coli. This finding led us to investigate whether the ability of PSIV IRES to initiate translation in E. coli is specific to this IRES and also to study features that allow this viral IRES to mediate prokaryotic translation initiation. We observed that certain IRESs may also possess the ability to initiate E. coli translation. Our results also indicated that the structural integrity of the PSIV IRES in translation in prokaryotes does not appear to be as critical as it is in eukaryotes. We also demonstrated that two regions of the PSIV IRES with complementarity to 16S ribosomal RNA are important for the ability of this IRES to initiate translation in E. coli.


Asunto(s)
Sitios Internos de Entrada al Ribosoma , Ribosomas , Secuencia de Bases , Ribosomas/metabolismo , Sitios Internos de Entrada al Ribosoma/genética , ARN Viral/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biosíntesis de Proteínas
6.
NAR Genom Bioinform ; 4(3): lqac058, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36004308

RESUMEN

The coronavirus disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prompted the development of diagnostic and therapeutic frameworks for timely containment of this pandemic. Here, we utilized our non-conventional computational algorithm, InSiPS, to rapidly design and experimentally validate peptides that bind to SARS-CoV-2 spike (S) surface protein. We previously showed that this method can be used to develop peptides against yeast proteins, however, the applicability of this method to design peptides against other proteins has not been investigated. In the current study, we demonstrate that two sets of peptides developed using InSiPS method can detect purified SARS-CoV-2 S protein via ELISA and Surface Plasmon Resonance (SPR) approaches, suggesting the utility of our strategy in real time COVID-19 diagnostics. Mass spectrometry-based salivary peptidomics shortlist top SARS-CoV-2 peptides detected in COVID-19 patients' saliva, rendering them attractive SARS-CoV-2 diagnostic targets that, when subjected to our computational platform, can streamline the development of potent peptide diagnostics of SARS-CoV-2 variants of concern. Our approach can be rapidly implicated in diagnosing other communicable diseases of immediate threat.

7.
Gene ; 831: 146549, 2022 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-35569766

RESUMEN

DNA repair defects are common in tumour cells and can lead to misrepair of double-strand breaks (DSBs), posing a significant challenge to cellular integrity. The overall mechanisms of DSB have been known for decades. However, the list of the genes that affect the efficiency of DSB repair continues to grow. Additional factors that play a role in DSB repair pathways have yet to be identified. In this study, we present a computational approach to identify novel gene functions that are involved in DNA damage repair in Saccharomyces cerevisiae. Among the primary candidates, GAL7, YMR130W, and YHI9 were selected for further analysis since they had not previously been identified as being active in DNA repair pathways. Originally, GAL7 was linked to galactose metabolism. YHI9 and YMR130W encode proteins of unknown functions. Laboratory testing of deletion strains gal7Δ, ymr130wΔ, and yhi9Δ implicated all 3 genes in Homologous Recombination (HR) and/or Non-Homologous End Joining (NHEJ) repair pathways, and enhanced sensitivity to DNA damage-inducing drugs suggested involvement in the broader DNA damage repair machinery. A subsequent genetic interaction analysis revealed interconnections of these three genes, most strikingly through SIR2, SIR3 and SIR4 that are involved in chromatin regulation and DNA damage repair network.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Daño del ADN/genética , Reparación del ADN/genética , Recombinación Homóloga , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética
8.
Mol Cell Biochem ; 477(11): 2643-2656, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35598219

RESUMEN

Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism enzyme phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5'-untranslated region (5'-UTR) of the PGM2 mRNA.


Asunto(s)
Cloruro de Litio , Proteínas de la Membrana , Peroxinas , Proteínas de Saccharomyces cerevisiae , Galactosa , Cloruro de Litio/farmacología , Proteínas de la Membrana/genética , Peroxinas/genética , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Regiones no Traducidas
9.
Int J Mol Sci ; 21(16)2020 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-32785068

RESUMEN

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of PGM2 mRNA in yeast.


Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Antimaníacos/farmacología , Cloruro de Litio/farmacología , Biosíntesis de Proteínas/genética , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Regiones no Traducidas 5' , Aminoacil-ARNt Sintetasas/genética , Antimaníacos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Cloruro de Litio/uso terapéutico , Organismos Modificados Genéticamente , Fosfoglucomutasa/antagonistas & inhibidores , Fosfoglucomutasa/metabolismo , ARN Helicasas/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genética
10.
PLoS One ; 15(7): e0235033, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32639961

RESUMEN

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.


Asunto(s)
Adenosina Trifosfatasas/genética , Antimaníacos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cloruro de Litio/farmacología , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoglucomutasa/genética , Biosíntesis de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
PLoS One ; 13(9): e0198704, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30231023

RESUMEN

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker's yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation.


Asunto(s)
Eliminación de Gen , Glutatión Peroxidasa/genética , Metales Pesados/metabolismo , Priones/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/genética , Regulación Fúngica de la Expresión Génica , Glutatión Peroxidasa/metabolismo , Inactivación Metabólica , Priones/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo
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