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1.
FEMS Yeast Res ; 17(3)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28449083

RESUMO

Yeast AP-1 transcription factor (Yap1p) and the enigmatic oxidoreductases Oye2p and Oye3p are involved in counteracting lipid oxidants and their unsaturated breakdown products. In order to uncover the response to linoleic acid hydroperoxide (LoaOOH) and the roles of Oye2p, Oye3p and Yap1p, we carried out proteomic analysis of the homozygous deletion mutants oye3Δ, oye2Δ and yap1Δ alongside the diploid parent strain BY4743. The findings demonstrate that deletion of YAP1 narrowed the response to LoaOOH, as the number of proteins differentially expressed in yap1Δ was 70% of that observed in BY4743. The role of Yap1p in regulating the major yeast peroxiredoxin Tsa1p was demonstrated by the decreased expression of Tsa1p in yap1Δ. The levels of Ahp1p and Hsp31p, previously shown to be regulated by Yap1p, were increased in LoaOOH-treated yap1Δ, indicating their expression is also regulated by another transcription factor(s). Relative to BY4743, protein expression differed in oye3Δ and oye2Δ under LoaOOH, underscored by superoxide dismutase (Sod1p), multiple heat shock proteins (Hsp60p, Ssa1p, and Sse1p), the flavodoxin-like protein Pst2p and the actin stabiliser tropomyosin (Tpm1p). Proteins associated with glycolysis were increased in all strains following treatment with LoaOOH. Together, the dataset reveals, for the first time, the yeast proteomic response to LoaOOH, highlighting the significance of carbohydrate metabolism, as well as distinction between the roles of Oye3p, Oye2p and Yap1p.


Assuntos
Regulação Fúngica da Expressão Gênica , Ácidos Linoleicos/farmacologia , Peróxidos Lipídicos/farmacologia , Oxidantes/farmacologia , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Flavodoxina/genética , Flavodoxina/metabolismo , Deleção de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Anotação de Sequência Molecular , Estresse Oxidativo , Oxirredutases/genética , Oxirredutases/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Proteoma/metabolismo , Proteômica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Biometals ; 25(3): 553-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22403011

RESUMO

Following our previous finding that the sulfhydryl-oxidising chemical diamide induced a marked elevation of cellular Al(3+) (Wu et al., Int J Mol Sci, 12:8119-8132, 2011), a further investigation into the underlying molecular mechanism was carried out, using the eukaryotic model organism Saccharomyces cerevisiae. The effects of non-toxic dose of diamide (0.8 mM) and a mild dose of aluminium sulphate (Al(3+)) (0.4 mM) were determined prior to the screening of gene deletion mutants. A total of 81 deletion mutants were selected for this study according to the available screening data against Al(3+) only (Kakimoto et al., BioMetals, 18: 467-474, 2005) and diamide only (Thorpe et al., Proc Natl Acad Sci USA, 101: 6564-6569, 2004). On the basis of our screening data and the cluster analysis, a cluster containing the gene deletions (rpe1∆, sec72∆, pdr5∆ and ric1∆) was found to be specifically sensitive to the mixture of diamide and Al(3+). However gnp1∆, mch5∆ and ccc1∆ mutants were resistant. Dithiothreitol (DTT) and ascorbate markedly reversed the diamide-induced Al(3+) toxicity. Inductively-coupled plasma optical emission spectrometry demonstrated that DTT reduced the intracellular Al(3+) content in diamide/Al(3+)-treated yeast cells six-fold compared to the non-DTT controls. These data together revealed that the pleiotropic drug resistance transporter (Pdr5p) and vacuolar/vesicular transport-related proteins (Ric1p and Sec72p) are the targets of diamide. A dysfunctional membrane-bound Pdr5p terminates the detoxification pathway for Al(3+) at the final step, leading to intracellular Al(3+) accumulation and hence toxicity. As Al(3+) toxicity has been a problem in agriculture and human health, this study has provided a significant step forward in understanding Al(3+) toxicity.


Assuntos
Alumínio/farmacologia , Dissulfetos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Diamida/farmacologia , Ditiotreitol/farmacologia , Saccharomyces cerevisiae/metabolismo
3.
FEMS Yeast Res ; 11(4): 379-87, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21375688

RESUMO

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.


Assuntos
Antioxidantes/análise , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Antioxidantes/farmacologia , Diamida/farmacologia , Espécies Reativas de Nitrogênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Saccharomyces cerevisiae/metabolismo , Superóxidos/farmacologia , Vitamina K 3/farmacologia
4.
Int J Mol Sci ; 12(9): 6089-103, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22016646

RESUMO

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.


Assuntos
Antioxidantes/metabolismo , Cerveja/análise , Armazenamento de Alimentos , Proteínas de Plantas/metabolismo , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Austrália , Compostos de Bifenilo/antagonistas & inibidores , Compostos de Bifenilo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/antagonistas & inibidores , Radicais Livres/metabolismo , Hordeum/metabolismo , Humanos , Picratos/antagonistas & inibidores , Picratos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Análise de Sequência de Proteína , Compostos de Sulfidrila/metabolismo , Temperatura , Fatores de Tempo
5.
Int J Mol Sci ; 12(11): 8119-32, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22174654

RESUMO

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al(3+)) level rose up to 50-fold after the diamide treatment. Cellular potassium (K(+)) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al(3+) accumulation was further validated by the enhanced Al(3+) uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al(3+) uptake, suggesting Al(3+)-specific transporters could be involved in Al(3+) uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.


Assuntos
Alumínio/metabolismo , Íons/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Derivados de Benzeno/farmacologia , Cobre/metabolismo , Cicloexanonas/farmacologia , Diamida/farmacologia , Peróxido de Hidrogênio/farmacologia , Ácidos Linoleicos/farmacologia , Peróxidos Lipídicos/farmacologia , Magnésio/metabolismo , Manganês/metabolismo , Modelos Moleculares , Oxidantes/farmacologia , Ácido Peroxinitroso/farmacologia , Potássio/metabolismo , Vitamina K 3/farmacologia
6.
Metallomics ; 9(3): 301-308, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28194465

RESUMO

Metal ions, biologically essential or toxic, are present in the surrounding environment of living organisms. Understanding their uptake, homeostasis or detoxification is critical in cell biology and human health. In this study, we investigated the role of protein kinase CK2 in metal toxicity using gene deletion strains of Saccharomyces cerevisiae against a panel of six metal ions. The deletion of CKA2, the yeast orthologue of mammalian CK2α', leads to a pronounced resistant phenotype against Zn2+ and Al3+, whilst the deletion of CKB1 or CKB2 results in tolerance to Cr6+ and As3+. The individual deletion mutants of CK2 subunits (CKA1, CKA2, CKB1 and CKB2) did not have any benefit against Co2+ and Cd2+. The metal ion content in the treated cells was then measured by inductively coupled plasma mass spectrometry. Two contrasting findings were obtained for the CKA2 deletion mutant (cka2Δ) against Al3+ or Zn2+. Upon exposure to Al3+, cka2Δ had markedly lower Al3+ content than the wild type and other CK2 mutants, congruous to the resistant phenotype of cka2Δ against Al3+, indicating that CKA2 is responsible for Al3+ uptake. Upon zinc exposure the same mutant showed similar Zn2+ content to the wild type and cka1Δ. Strikingly, the selective inhibitor of CK2 TBB (4,5,6,7-tetrabromo-1H-benzotriazole) abolished the resistant phenotype of cka2Δ against Zn2+. Hence, the CK2 subunit CKA1 plays a key role in Zn2+ sequestration of the cell. Given that both zinc and CK2 are implicated in cancer development, the findings herein are of significance to cancer research and anticancer drug development.


Assuntos
Caseína Quinase II/genética , Deleção de Genes , Intoxicação por Metais Pesados/etiologia , Metais/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Intoxicação por Metais Pesados/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
7.
Metallomics ; 8(5): 542-50, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27146641

RESUMO

Chromium toxicity is increasingly relevant to living organisms such as humans, due to the environmental contamination of chromium and the application of stainless steel-based medical devices like hip prostheses. Despite the investigations in past years, the molecular details for chromium toxicity remain to be delineated. In this study, we seek to gain insights into the molecular aspects of chromium toxicity by screening a genome-wide deletion set of individual genes in Saccharomyces cerevisiae against hexavalent chromium [Cr(vi)] using chromium trioxide. From the primary data collected in this study, two lists of deletion mutants in response to Cr(vi) exposure were obtained, one for the sensitive phenotype and the other for the resistant phenotype. The functional analysis of the genes corresponding to the sensitive mutants reveals the key features of Cr(vi) toxicity, which include genotoxicity, protein damage, disruption of energy and sulfur metabolisms. DNA repair, ubiquitination-mediated protein degradation, iron homeostasis and growth attenuation are the intrinsic facets of the cell's detoxification mechanisms. Protein kinase CK2 is, for the first time, found to be involved in regulating chromium toxicity by reducing the uptake of Cr(vi). Taken together, the findings provide meaningful details into the basic understanding of chromium toxicity in terms of its uptake, modes of action, cellular detoxification and molecular regulatory mechanisms.


Assuntos
Cromo/toxicidade , Deleção de Genes , Genes Fúngicos , Saccharomyces cerevisiae/efeitos dos fármacos , Dano ao DNA , Saccharomyces cerevisiae/genética
8.
Metallomics ; 8(2): 228-35, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26688044

RESUMO

Arsenic is omnipresent in soil, air, food and water. Chronic exposure to arsenic is a serious problem to human health. In-depth understanding of this metalloid's toxicity is a fundamental step towards development of arsenic-free foods and measures for bioremediation. By screening the complete set of gene deletion mutants (4873) of Saccharomyces cerevisiae, this study uncovered 75 sensitive and 39 resistant mutants against arsenite [As(III)]. Functional analysis of the corresponding genes revealed the molecular details for its uptake, toxicity and detoxification. On the basis of the hypersensitivity of yap3Δ, the transcription factor, Yap3p, is for the first time linked to the cell's detoxification against As(III). Apart from confirming the previously described role of the mitogen-activated protein kinase (MAPK) Hog1 pathway in combating arsenic toxicity, the results show that the regulatory subunits (Ckb1p and Ckb2p) of protein kinase CK2 are also involved in the process, suggesting possible crosstalk between the two key protein kinases. The sensitivity to As(III) conferred by deletion of the genes involved in protein degradation and chromatin remodelling demonstrates protein damage is the key mode of toxicity for the metalloid. Furthermore, the resistant phenotype of fps1Δ, snf3Δ and pho81Δ against As(III) links arsenic uptake with the corresponding plasma membrane-bound transporters-aquaglyceroporin (Fps1p), hexose (Snf3p) and phosphate transporters. The molecular details obtained in this screen for As(III) uptake, detoxification and toxicity provide the basis for future investigations into arsenic-related problems in the environment, agriculture and human health.


Assuntos
Arsênio/toxicidade , Poluentes Ambientais/toxicidade , Genoma Fúngico/efeitos dos fármacos , Saccharomyces cerevisiae , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
9.
Metallomics ; 6(8): 1558-64, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24926745

RESUMO

Genome-wide screening using gene deletion mutants has been widely carried out with numerous toxicants including oxidants and metal ions. The focus of such studies usually centres on identifying sensitive phenotypes against a given toxicant. Here, we screened the complete collection of yeast gene deletion mutants (5047) with increasing concentrations of aluminium sulphate (0.4, 0.8, 1.6 and 3.2 mM) in order to discover aluminium (Al(3+)) tolerant phenotypes. Fifteen genes were found to be associated with Al(3+) transport because their deletion mutants exhibited Al(3+) tolerance, including lem3Δ, hal5Δ and cka2Δ. Deletion of CKA2, a catalytic subunit of tetrameric protein kinase CK2, gives rise to the most pronounced resistance to Al(3+) by showing significantly higher growth compared to the wild type. Functional analysis revealed that both molecular regulation and endocytosis are involved in Al(3+) transport for yeast. Further investigations were extended to all the four subunits of CK2 (CKA1, CKA2, CKB1 and CKB2) and the other 14 identified mutants under a spectrum of metal ions, including Al(3+), Zn(2+), Mn(2+), Fe(2+), Fe(3+), Co(3+), Ga(3+), Cd(2+), In(3+), Ni(2+) and Cu(2+), as well as hydrogen peroxide and diamide, in order to unravel cross-tolerance amongst metal ions and the effect of the oxidants. Finally, the implication of the findings in Al(3+) transport for the other species like plants and humans is discussed.


Assuntos
Alumínio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Íons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
10.
Metallomics ; 5(8): 1068-75, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23832094

RESUMO

Formation of non-native disulfide bonds within or between proteins can lead to protein misfolding and disruption to cellular metabolism. Such a process is defined as disulfide stress. A marked effect of disulfide stress in cells is the elevated accumulation of the intracellular aluminium ion (Al(3+)) accompanied by increased cytotoxicity. To gain an in-depth understanding of the underlying molecular mechanism for disulfide stress-induced aluminium toxicity, the complete set of Saccharomyces cerevisiae deletion mutants (5047) was screened in this study simultaneously with a combination of the two stressors, diamide and Al(3+). The combined treatment of a benign concentration of diamide (0.8 mM) with a sublethal concentration of aluminium sulfate (0.4 mM) revealed 494 sensitive deletion mutants, distinct from those found when either of the single stressors (0.8 mM diamide or 0.4 mM aluminium sulfate) was used. Hierarchical clustering and functional analyses of the 494 mutants sensitive to the dual stressors indicated a significant enrichment in the genes involved in cell wall homeostasis, signaling cascades, secretory transport machinery and detoxification. The results highlight the process of maintaining cell wall integrity as the central response to the combined exposure of diamide and Al(3+), which is mediated by the signaling pathways and transcription activation via Rlm1p and Swi6p for biosynthesis of the essential cell wall components such as glucan and chitin. Sensitivity of mutants associated with endoplasmic reticulum (ER), vesicle and vacuole functions demonstrates that secretory machinery is essential for surviving the stress conditions, probably due to their roles in transporting polysaccharides to the cell wall and detoxification of accumulated Al(3+). Finally, the phenotype of 100 previously uncharacterized genes against the dual stressors will contribute to their eventual functional annotation.


Assuntos
Alumínio/química , Dissulfetos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Quitina/química , Diamida/química , Retículo Endoplasmático/metabolismo , Deleção de Genes , Genoma Fúngico , Glucanos/química , Íons , Proteínas de Domínio MADS/metabolismo , Mutação , Fenótipo , Polissacarídeos/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
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