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1.
FASEB J ; 35(9): e21778, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383971

RESUMO

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.


Assuntos
Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Glicerol/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Estilbenos/farmacologia , Transcrição Genética/efeitos dos fármacos , beta-Glucanas/farmacologia , Caspofungina/farmacologia , Parede Celular/genética , Parede Celular/metabolismo , Quitina/farmacologia , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Concentração Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Genética/genética
2.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199487

RESUMO

Phenolic compounds are thought to be important to prevent neurodegenerative diseases (ND). Parkinson's Disease (PD) is a neurodegenerative disorder known for its typical motor features, the deposition of α-synuclein (αsyn)-positive inclusions in the brain, and for concomitant cellular pathologies that include oxidative stress and neuroinflammation. Neuroprotective activity of fisetin, a dietary flavonoid, was evaluated against main hallmarks of PD in relevant cellular models. At physiologically relevant concentrations, fisetin protected SH-SY5Y cells against oxidative stress overtaken by tert-butyl hydroperoxide (t-BHP) and against methyl-4-phenylpyridinuim (MPP+)-induced toxicity in dopaminergic neurons, the differentiated Lund human Mesencephalic (LUHMES) cells. In this cellular model, fisetin promotes the increase of the levels of dopamine transporter. Remarkably, fisetin reduced the percentage of cells containing αsyn inclusions as well as their size and subcellular localization in a yeast model of αsyn aggregation. Overall, our data show that fisetin exerts modulatory activities toward common cellular pathologies present in PD; remarkably, it modulates αsyn aggregation, supporting the idea that diets rich in this compound may prove beneficial.


Assuntos
Butiratos/efeitos adversos , Flavonóis/farmacologia , Doença de Parkinson/metabolismo , Piperidinas/efeitos adversos , alfa-Sinucleína/metabolismo , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Modelos Biológicos , Estresse Oxidativo , Doença de Parkinson/tratamento farmacológico , Agregados Proteicos/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , terc-Butil Hidroperóxido/metabolismo
3.
Mol Genet Genomics ; 296(5): 1135-1145, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196769

RESUMO

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.


Assuntos
Proteínas Fúngicas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Magnaporthe/enzimologia , Dioxóis/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201004

RESUMO

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.


Assuntos
Cádmio/toxicidade , Parede Celular/química , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Fosforilação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais
5.
Plant Cell Rep ; 40(9): 1751-1772, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34173048

RESUMO

KEY MESSAGE: Early induction of OsFEX was insufficient for fluoride adaptation in IR-64. Overexpression of OsFEX in yeast and Nicotiana benthamiana enhanced fluoride tolerance. The present study delineates the regulation of fluoride exporter (FEX) in the fluoride-sensitive rice cultivar, IR-64 and its efficacy in generating high fluoride tolerance in transgenic Nicotiana benthamiana. Gene and protein expression profiling revealed that OsFEX exhibited early induction during fluoride stress in the vegetative and reproductive tissues of IR-64, although the expression was suppressed upon prolonged stress treatment. Analysis of OsFEX promoter in transgenic N. benthamiana, using ß-glucuronidase reporter assay confirmed its early inducible nature, since the reporter expression and activity peaked at 12 h of NaF stress, after which it was lowered. OsFEX expression was up regulated in the presence of gibberellic acid (GA) and melatonin, while it was suppressed by abscisic acid (ABA). Complementation of ΔFEX1ΔFEX2 yeast mutants with OsFEX enabled high fluoride tolerance, thus validating the functional efficiency of the transgene. Bioassay of transgenic N. benthamiana lines, expressing OsFEX either under its own promoter or under CaMV35S promoter, established that constitutive overexpression, rather than early induction of OsFEX was essential and crucial for generating fluoride tolerance in the transgenics. Overall, the suppression of OsFEX in the later growth phases of stressed IR-64 due to enhanced ABA conservation and lowered synthesis of GA, as supported by the application of the respective phytohormone biosynthetic inhibitors, such as sodium tungstate and paclobutrazol, accounted for the fluoride-hyperaccumulative nature of the rice cultivar.


Assuntos
Fluoretos/toxicidade , Oryza/efeitos dos fármacos , Oryza/fisiologia , Proteínas de Plantas/genética , Tabaco/genética , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fluoretos/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Giberelinas/farmacologia , Proteínas de Membrana/genética , Microrganismos Geneticamente Modificados , Mutação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia
6.
Food Chem ; 362: 130240, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34119950

RESUMO

The alleviating role of starch derivatives on the quality deterioration of frozen steamed bread dough was investigated in terms of derivative structure, the bread characteristics and dough properties including freezable water contents, yeast activity as well as dough viscoelasticity. The addition of starch derivatives including short-clustered maltodextrin (SCMD), DE2 maltodextrin (MD) and pregelatinized starch (PGS) significantly increased the specific volume and decreased the hardness of steamed bread compared with Control bread after 8-week frozen storage. Lower freezable water content was found in PGS dough than SCMD dough, which was consistent with the results of water absorption index of starch derivatives. The analysis of dough gassing rate and yeast survival ratio demonstrated SCMD could provide more cryoprotection for yeast cells. Meanwhile, a higher elastic module and a more continuous gluten-network structure of SCMD dough were found after 8-week frozen storage. These results indicated starch derivatives especially SCMD were promising to be used as the alternative improvers in frozen dough production.


Assuntos
Pão/análise , Pão/normas , Qualidade dos Alimentos , Congelamento , Amido/farmacologia , Tecnologia de Alimentos , Glutens/química , Saccharomyces cerevisiae/efeitos dos fármacos , Amido/química , Vapor , Viscosidade/efeitos dos fármacos , Água/química , Fermento Seco/análise
7.
FEBS Lett ; 595(14): 1886-1901, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096057

RESUMO

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.


Assuntos
Actinas/genética , Substituição de Aminoácidos , Ácido Aspártico/química , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Actinas/química , Actinas/metabolismo , Alanina/química , Alanina/metabolismo , Alelos , Ácido Aspártico/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Compostos de Sulfonilureia/farmacologia
8.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067304

RESUMO

Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metanol/análogos & derivados , Compostos Organosselênicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Metanol/farmacologia , Chaperonas Moleculares/metabolismo , Oxirredução/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071169

RESUMO

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.


Assuntos
Antifúngicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Antifúngicos/química , Cálcio , Farmacorresistência Fúngica/genética , Homeostase/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Nucleossomos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Transdução de Sinais , ATPases Vacuolares Próton-Translocadoras
10.
Nat Commun ; 12(1): 2803, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990576

RESUMO

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.


Assuntos
Antibacterianos/farmacologia , Macrolídeos/farmacologia , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Sítios de Ligação , Microscopia Crioeletrônica , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Humanos , Macrolídeos/química , Modelos Moleculares , Mutação , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Relação Estrutura-Atividade
11.
Food Chem ; 361: 130025, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34029908

RESUMO

The relative concentration of available inorganic elements is critical for yeast growth and metabolism and has potential to be a tool leading to directed yeast flavour formation during fermentation. This study investigates the influence of essential inorganic elements during alcoholic fermentation of brewers wort, fermented using three independent yeast strains, Saccharomyces pastorianus W34/70, and Saccharomyces cerevisiae strains M2 and NCYC2592 under a range of conditions replicated for each yeast strain. 10 treatments were applied: 1 control and 9 inorganic supplementations: standard brewers wort, ammonia-nitrogen, inorganic phosphate, potassium, magnesium, copper, zinc, iron, manganese and a composite mixture, Twenty-five chemical markers were evaluated by HPLC (ethanol, glycerol), and GC-MS (aroma). There was a significant change in volatile aroma compounds during fermentation, which was more prominent when supplemented with ammonia nitrogen, inorganic phosphate, potassium or magnesium (P < 0.05). Heavy metal ions mostly had a negative effect on the flavour formation.


Assuntos
Cerveja/microbiologia , Metais/farmacologia , Saccharomyces/metabolismo , Cerveja/análise , Cromatografia Líquida de Alta Pressão , Etanol/metabolismo , Fermentação/efeitos dos fármacos , Microbiologia de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Glicerol/metabolismo , Metais/metabolismo , Odorantes , Potássio/metabolismo , Potássio/farmacologia , Saccharomyces/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo
12.
Appl Environ Microbiol ; 87(13): e0310020, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893111

RESUMO

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is a vital cell factory, but more diverse cell factories are essential for the sustainable use of natural resources. Here, we benchmarked nonconventional yeasts Kluyveromyces marxianus and Rhodotorula toruloides against S. cerevisiae strains CEN.PK and W303 for their responses to potassium and sodium salt stress. We found an inverse relationship between the maximum growth rate and the median cell volume that was responsive to salt stress. The supplementation of K+ to CEN.PK cultures reduced Na+ toxicity and increased the specific growth rate 4-fold. The higher K+ and Na+ concentrations impaired ethanol and acetate metabolism in CEN.PK and acetate metabolism in W303. In R. toruloides cultures, these salt supplementations induced a trade-off between glucose utilization and cellular aggregate formation. Their combined use increased the beta-carotene yield by 60% compared with that of the reference. Neural network-based image analysis of exponential-phase cultures showed that the vacuole-to-cell volume ratio increased with increased cell volume for W303 and K. marxianus but not for CEN.PK and R. toruloides in response to salt stress. Our results provide insights into common salt stress responses in yeasts and will help design efficient bioprocesses. IMPORTANCE Characterization of microbial cell factories under industrially relevant conditions is crucial for designing efficient bioprocesses. Salt stress, typical in industrial bioprocesses, impinges upon cell volume and affects productivity. This study presents an open-source neural network-based analysis method to evaluate volumetric changes using yeast optical microscopy images. It allows quantification of cell and vacuole volumes relevant to cellular physiology. On applying salt stress in yeasts, we found that the combined use of K+ and Na+ improves the cellular fitness of Saccharomyces cerevisiae strain CEN.PK and increases the beta-carotene productivity in Rhodotorula toruloides, a commercially important antioxidant and a valuable additive in foods.


Assuntos
Kluyveromyces/efeitos dos fármacos , Potássio/farmacologia , Rhodotorula/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Salino , Sódio/farmacologia , Acetatos/metabolismo , Etanol/metabolismo , Glucose/metabolismo , Kluyveromyces/metabolismo , Rhodotorula/metabolismo , Saccharomyces cerevisiae/metabolismo
13.
Chem Biodivers ; 18(6): e2100054, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33915032

RESUMO

Eugenia copacabanensis and Myrciaria tenella are present in restingas of the Atlantic Forest, but little information is available about their chemical and biological potential. In this context, the hexane, dichloromethane, ethyl acetate and butanol fractions from the leaves of methanolic extract were analyzed by GC/MS and HPLC-DAD and the antioxidant potential was determined by DPPH and ABTS assays and using a Saccharomyces cerevisiae model. Dereplication allowed the identification of 68 compounds, 42 and 41 of which, respectively, are first reported here for E. copacabanensis and M. tenella. In vivo results revealed that the ethyl acetate and butanol fractions showed expressive antioxidant protection in the BY4741 and Δgsh1 strains, with greater impact on glutathione-deficient cells. With a high diversity of phenolic compounds, these polar fractions of E. copacabanensis and M. tenella leaves are potential protectors against intracellular oxidative stress.


Assuntos
Antifúngicos/farmacologia , Antioxidantes/farmacologia , Eugenia/química , Myrtaceae/química , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Antifúngicos/análise , Antioxidantes/análise , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Modelos Biológicos , Compostos Fitoquímicos/análise , Picratos/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ácidos Sulfônicos/antagonistas & inibidores
14.
Genes (Basel) ; 12(3)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802854

RESUMO

The primary focus of our research was to obtain global gene expression data in baker's yeast exposed to sub-lethal doses of quantum dots (QDs), such as green-emitting CdSe/ZnS and InP/ZnS, to reveal novel insights on their unique mechanisms of toxicity. Despite their promising applications, their toxicity and long-lasting effects on the environment are not well understood. To assess toxicity, we conducted cell viability assays, ROS detection assays, and assessed their effects on the trafficking of Vps10-GFP toward the trans-Golgi network with confocal microscopy. Most notably, we used RNA-sequencing (RNA-seq) to obtain gene expression profiles and gene identities of differentially expressed genes (DEGs) in QD-treated yeast. We found CdSe/ZnS QDs significantly altered genes implicated in carboxylic acid, amino acid, nitrogen compounds, protein metabolic processes, transmembrane transport, cellular homeostasis, cell wall organization, translation, and ribosomal biogenesis. Additionally, we found InP/ZnS QDs to alter genes associated with oxidation-reduction, transmembrane transport, metal ion homeostasis, cellular component organization, translation, and protein and nitrogen compound metabolic processes. Interestingly, we observed an increase in reactive oxygen species (ROS) in CdSe/ZnS-treated cells and a decrease in ROS levels in InP/ZnS-treated cells. Nevertheless, we concluded that both QDs modestly contributed cytotoxic effects on the budding yeast.


Assuntos
Perfilação da Expressão Gênica/métodos , Pontos Quânticos/toxicidade , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Compostos de Cádmio/toxicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Índio/toxicidade , Viabilidade Microbiana/efeitos dos fármacos , Fosfinas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Compostos de Selênio/toxicidade , Análise de Sequência de RNA , Sulfetos/toxicidade , Compostos de Zinco/toxicidade
15.
Plant Cell ; 33(2): 420-438, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33866370

RESUMO

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Boro/farmacologia , Proteólise/efeitos dos fármacos , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Antiporters/química , Proteínas de Arabidopsis/química , Sítios de Ligação , Testes Genéticos , Proteínas de Fluorescência Verde/metabolismo , Lisina/metabolismo , Modelos Biológicos , Poliubiquitina/metabolismo , Transporte Proteico/efeitos dos fármacos , Prótons , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Ubiquitinação/efeitos dos fármacos , Vacúolos/metabolismo
16.
J Vis Exp ; (170)2021 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-33900287

RESUMO

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker's yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.


Assuntos
Genoma Fúngico , Genoma Mitocondrial , RNA Mensageiro/genética , RNA Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cicloeximida/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Coloração e Rotulagem/métodos
17.
BMC Cancer ; 21(1): 356, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33823841

RESUMO

BACKGROUND: Evidence bearing on the role of statins in the prevention and treatment of cancer is confounded by the diversity of statins, chemotherapeutic agents and cancer types included in the numerous published studies; consequently, the adjunctive value of statins with chemotherapy remains uncertain. METHODS: We assayed lovastatin in combination with each of ten commonly prescribed chemotherapy drugs in highly reproducible in vitro assays, using a neutral cellular substrate, Saccharomyces cerevisiae. Cell density (OD600) data were analyzed for synergism and antagonism using the Loewe additivity model implemented with the Combenefit software. RESULTS: Four of the ten chemotherapy drugs - tamoxifen, doxorubicin, methotrexate and rapamycin - exhibited net synergism with lovastatin. The remaining six agents (5-fluorouracil, gemcitabine, epothilone, cisplatin, cyclophosphamide and etoposide) compiled neutral or antagonistic scores. Distinctive patterns of synergism and antagonism, often coexisting within the same concentration space, were documented with the various combinations, including those with net synergism scores. Two drug pairs, lovastatin combined with tamoxifen or cisplatin, were also assayed in human cell lines as proof of principle. CONCLUSIONS: The synergistic interactions of tamoxifen, doxorubicin, methotrexate and rapamycin with lovastatin - because they suggest the possibility of clinical utility - merit further exploration and validation in cell lines and animal models. No less importantly, strong antagonistic interactions between certain agents and lovastatin argue for a cautious, data-driven approach before adding a statin to any chemotherapeutic regimen. We also urge awareness of adventitious statin usage by patients entering cancer treatment protocols.


Assuntos
Anticolesterolemiantes/uso terapêutico , Antagonismo de Drogas , Sinergismo Farmacológico , Lovastatina/uso terapêutico , Saccharomyces cerevisiae/efeitos dos fármacos , Anticolesterolemiantes/farmacologia , Humanos , Lovastatina/farmacologia , Preparações Farmacêuticas
18.
Nat Commun ; 12(1): 2452, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907191

RESUMO

The cell cycle is the process by which eukaryotic cells replicate. Yeast cells cycle asynchronously with each cell in the population budding at a different time. Although there are several experimental approaches to synchronise cells, these usually work only in the short-term. Here, we build a cyber-genetic system to achieve long-term synchronisation of the cell population, by interfacing genetically modified yeast cells with a computer by means of microfluidics to dynamically change medium, and a microscope to estimate cell cycle phases of individual cells. The computer implements a controller algorithm to decide when, and for how long, to change the growth medium to synchronise the cell-cycle across the population. Our work builds upon solid theoretical foundations provided by Control Engineering. In addition to providing an avenue for yeast cell cycle synchronisation, our work shows that control engineering can be used to automatically steer complex biological processes towards desired behaviours similarly to what is currently done with robots and autonomous vehicles.


Assuntos
Ciclo Celular/genética , Ciclinas/genética , Retroalimentação Fisiológica , GTP Fosfo-Hidrolases/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Algoritmos , Automação Laboratorial , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Ciclinas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Int J Mol Sci ; 22(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923309

RESUMO

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.


Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Ensaios de Triagem em Larga Escala/métodos , Translocases Mitocondriais de ADP e ATP/genética , Doenças Mitocondriais/tratamento farmacológico , Mutação , Preparações Farmacêuticas/administração & dosagem , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , DNA Mitocondrial/genética , Genes Dominantes , Humanos , Doenças Mitocondriais/genética , Oftalmoplegia/tratamento farmacológico , Oftalmoplegia/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
20.
PLoS One ; 16(3): e0235303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33730086

RESUMO

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.


Assuntos
Etídio/farmacologia , Metanossulfonato de Metila/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Canavanina/farmacologia , Meios de Cultura/química , DNA Fúngico/química , DNA Fúngico/metabolismo , Testes de Mutagenicidade/instrumentação , Testes de Mutagenicidade/métodos , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
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