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2.
PLoS Biol ; 18(8): e3000757, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32833957

RESUMO

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.


Assuntos
Adaptação Fisiológica/genética , Lisina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Nutrientes/farmacologia , Saccharomyces cerevisiae/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Evolução Biológica , Glucose/metabolismo , Glucose/farmacologia , Lisina/deficiência , Redes e Vias Metabólicas/genética , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Nutrientes/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirolimo/farmacologia , Estresse Fisiológico
3.
Chem Biol Interact ; 330: 109231, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853594

RESUMO

Hetero mononuclear rhenium(I) metal complexes (I-V) using different substituted indole-pyrazoline based ligands were synthesized and characterized by spectroscopic and analytical methods. The binding of the rhenium complexes to Herring sperm DNA was monitored by UV spectroscopy, viscosity measurements, and molecular docking studies; groove binding was suggested as the most possible mode and the DNA-binding constants of the complexes were evaluated. In vivo and in vitro cytotoxicity of compounds were evaluated against the brine shrimp and S. cerevisiae cells. An antimicrobial study was carried out by estimating MIC (Minimum Inhibitory Concentration) against two Gram-positive and three Gram-negative bacteria. All synthesized complexes are biologically more active than the corresponding ligands. The anti-proliferation activity of complexes was evaluated on MCF-7, HCT116, and A549 cancer cells by MTT assay. The toxicity profile of synthesized compounds was confirmed by H2O2 production by reactive oxygen species. The increased concentration of lipid peroxidation end products increased free radicals, which enhancing the oxidative stress level in living organisms and results in cell death.


Assuntos
Complexos de Coordenação/farmacologia , Indóis/química , Pirazóis/química , Rênio/química , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Artemia/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Citotoxinas/toxicidade , DNA/metabolismo , Humanos , Ligantes , Peroxidação de Lipídeos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos
4.
Ecotoxicol Environ Saf ; 205: 111131, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827964

RESUMO

Arsenic (As) is one of the most toxic contaminants to food crops, and as such, decreasing crops uptake and accumulation of As cannot be overemphasized. Here, we characterized a functional wheat NIP2;1 homolog of the As transporter, TaNIP2;1. TaNIP2;1 expression was suppressed by arsenite (As(III)) in wheat. Ectopic expression of TaNIP2;1 in the Δfps1 yeast mutant enhanced yeast sensitivity towards As(III). Conversely, the elevated expression of TaNIP2;1 in Δacr3 mutants decreased yeast sensitivity to arsenate (As(V)), demonstrating that TaNIP2;1 showed both influx and efflux transport activities for As(III) in yeasts. This is further supported by increased As concentration in the yeast cells that overproduce TaNIP2;1 in Δfps1, while As concentration decreased in Δacr3. Furthermore, ectopic expression of TaNIP2;1 in Arabidopsis confirmed that TaNIP2;1 can transport As into plants, as supported by increased sensitivity to and uptake of As(III). No change in plant sensitivity was found to Cu(II), Cd(II), Zn(II) or Ni(II), indicating that transport activity of TaNIP2;1 is specific for As(III). Taken together, our data show that TaNIP2;1 may be involved in As(III) transportation in plants. This finding reveals a functional gene that can be manipulated to reduce As content in wheat.


Assuntos
Aquagliceroporinas/genética , Arabidopsis/efeitos dos fármacos , Arsenitos/toxicidade , Expressão Ectópica do Gene/efeitos dos fármacos , Poluentes do Solo/toxicidade , Triticum/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Aquagliceroporinas/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arsenitos/metabolismo , Bioacumulação , Transporte Biológico , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Poluentes do Solo/metabolismo , Triticum/genética , Triticum/metabolismo
5.
PLoS One ; 15(7): e0235033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639961

RESUMO

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.


Assuntos
Adenosina Trifosfatases/genética , Antimaníacos/farmacologia , Cloreto de Lítio/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fosfoglucomutase/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
6.
Mol Cell ; 79(4): 588-602.e6, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32615089

RESUMO

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.


Assuntos
Pegada de DNA/métodos , Genoma Fúngico , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/metabolismo , Regiões 3' não Traduzidas , Anisomicina/farmacologia , Códon de Terminação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mutação , Fosforilação , Estabilidade de RNA , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
7.
PLoS Genet ; 16(7): e1008484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673313

RESUMO

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do Genoma
8.
Environ Health Prev Med ; 25(1): 30, 2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680455

RESUMO

Methylmercury is an environmental pollutant that causes neurotoxicity. Recent studies have reported that the ubiquitin-proteasome system is involved in defense against methylmercury toxicity through the degradation of proteins synthesizing the pyruvate. Mitochondrial accumulation of pyruvate can enhance methylmercury toxicity. In addition, methylmercury exposure induces several immune-related chemokines, specifically in the brain, and may cause neurotoxicity. This summary highlights several molecular mechanisms of methylmercury-induced neurotoxicity.


Assuntos
Quimiocinas/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Neurotoxinas/toxicidade , Proteólise/efeitos dos fármacos , Animais , Quimiocinas/metabolismo , Humanos , Camundongos , Ratos , Saccharomyces cerevisiae/efeitos dos fármacos
9.
Nat Commun ; 11(1): 3664, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694532

RESUMO

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/toxicidade , Taxa de Mutação , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Mutagênese , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Nucleic Acids Res ; 48(10): 5407-5425, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356874

RESUMO

Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3' end processing machinery to the vicinity of pA sites.


Assuntos
Histona-Lisina N-Metiltransferase/fisiologia , Metiltransferases/fisiologia , Poliadenilação , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Cromatina/química , Cromatina/efeitos dos fármacos , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas , Metiltransferases/genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
11.
Nucleic Acids Res ; 48(11): 6210-6222, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32365182

RESUMO

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.


Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ricina/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endorribonucleases/farmacologia , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ricina/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento
12.
Nat Commun ; 11(1): 1780, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286267

RESUMO

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Concentração Inibidora 50 , Espectrometria de Massas , Proteínas de Protozoários/metabolismo , Pirazóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Via Secretória/efeitos dos fármacos
13.
J Biosci Bioeng ; 130(2): 142-148, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32327386

RESUMO

As a large group of natural product with significant biological activities, triterpenoid secretion is of particular importance towards its bioproduction. Due to the lack of specific transporters, most triterpenoids are naturally accumulated inside the cells. In this study, by taking an antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid (GA-HLDOA) as example, we discovered that addition of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) or 2,6-dimethyl-ß-cyclodextrin (DM-ß-CD) enable the fast and sufficient secretion of GA-HLDOA by the recombinant Saccharomyces cerevisiae strain as constructed in our previous study. In addition, these cyclodextrins (CDs) could not enter into cells, while no significant change of the cell membrane fluidity was observed after CDs treatment. This discovery provides a potential generally applicable method for triterpenoid secretion.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Triterpenos/metabolismo , beta-Ciclodextrinas/farmacologia , Ciclodextrinas/farmacologia , Microbiologia Industrial , Saccharomyces cerevisiae/metabolismo
14.
PLoS One ; 15(3): e0229796, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134970

RESUMO

Chaperones and autophagy are components of the protein quality control system that contribute to the management of proteins that are misfolded and aggregated. Here, we use yeast prions, which are self-perpetuating aggregating proteins, as a means to understand how these protein quality control systems influence aggregate loss. Chaperones, such as Hsp104, fragment prion aggregates to generate more prion seeds for propagation. While much is known about the role of chaperones, little is known about how other quality control systems contribute to prion propagation. We show that the aprotic solvent dimethyl sulfoxide (DMSO) cures a range of [PSI+] prion variants, which are related to several misfolded aggregated conformations of the Sup35 protein. Our studies show that DMSO-mediated curing is quicker and more efficient than guanidine hydrochloride, a prion curing agent that inactivates the Hsp104 chaperone. Instead, DMSO appears to induce Hsp104 expression. Using the yTRAP system, a recently developed transcriptional reporting system for tracking protein solubility, we found that DMSO also rapidly induces the accumulation of soluble Sup35 protein, suggesting a potential link between Hsp104 expression and disassembly of Sup35 from the prion aggregate. However, DMSO-mediated curing appears to also be associated with other quality control systems. While the induction of autophagy alone does not lead to curing, we found that DMSO-mediated curing is dramatically impaired in autophagy related (atg) gene mutants, suggesting that other factors influence this DMSO mechanism of curing. Our data suggest that DMSO-mediated curing is not simply dependent upon Hsp104 overexpression alone, but may further depend upon other aspects of proteostasis.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Dimetil Sulfóxido/farmacologia , Proteínas de Choque Térmico/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Mutação , Fatores de Terminação de Peptídeos/antagonistas & inibidores , Príons/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
15.
Biochim Biophys Acta Biomembr ; 1862(8): 183255, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32145284

RESUMO

The plant defensin HsAFP1 is characterized by broad-spectrum antifungal activity and induces apoptosis in Candida albicans. In this study, we performed a transcriptome analysis on C. albicans cultures treated with HsAFP1 to gain further insight in the antifungal mode of action of HsAFP1. Various genes coding for cell surface proteins, like glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in cation homeostasis, autophagy and in cell cycle were differentially expressed upon HsAFP1 treatment. The biological validation of these findings was performed in the model yeast Saccharomyces cerevisiae. To discriminate between events linked to HsAFP1's antifungal activity and those that are not, we additionally used an inactive HsAFP1 mutant. We demonstrated that (i) HsAFP1-resistent S. cerevisiae mutants that are characterized by a defect in processing GPI-anchors are unable to internalize HsAFP1, and (ii) moderate doses (FC50, fungicidal concentration resulting in 50% killing) of HsAFP1 induce autophagy in S. cerevisiae, while high HsAFP1 doses result in vacuolar dysfunction. Vacuolar function is an important determinant of replicative lifespan (RLS) under dietary restriction (DR). In line, HsAFP1 specifically reduces RLS under DR. Lastly, (iii) HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. However, the latter HsAFP1-induced event is not linked to its antifungal activity, as the inactive HsAFP1 mutant also impairs the G2/M phase. In conclusion, we demonstrated that GPI-anchored proteins are involved in HsAFP1's internalization, and that HsAFP1 induces autophagy, vacuolar dysfunction and impairment of the cell cycle. Collectively, all these data provide novel insights in the mode of action of HsAFP1 as well as in S. cerevisiae tolerance mechanisms against this peptide.


Assuntos
Autofagia/efeitos dos fármacos , Defensinas/química , Heuchera/química , Saccharomyces cerevisiae/efeitos dos fármacos , Antifúngicos/química , Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Defensinas/genética , Defensinas/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/genética
16.
Cell Physiol Biochem ; 54(2): 211-229, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32100973

RESUMO

BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Cobre/farmacologia , DNA Mitocondrial/metabolismo , Dimerização , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
17.
Nat Cell Biol ; 22(2): 159-166, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029894

RESUMO

Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia/genética , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Citoplasma/metabolismo , Glucose/farmacologia , Complexos Multiproteicos/metabolismo , Nitrogênio/farmacologia , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia
18.
Nat Commun ; 11(1): 867, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054832

RESUMO

Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-ß peptides (Aß) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aß42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aß42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aß42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Mononucleotídeo de Flavina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Genes Sintéticos , Genoma Fúngico , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Genéticos , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Dobramento de Proteína , Proteólise , RNA-Seq , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
19.
Sci Adv ; 6(3): eaax8582, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32010767

RESUMO

This work establishes a means to exploit genetic networks to create living synthetic composites that change shape in response to specific biochemical or physical stimuli. Baker's yeast embedded in a hydrogel forms a responsive material where cellular proliferation leads to a controllable increase in the composite volume of up to 400%. Genetic manipulation of the yeast enables composites where volume change on exposure to l-histidine is 14× higher than volume change when exposed to d-histidine or other amino acids. By encoding an optogenetic switch into the yeast, spatiotemporally controlled shape change is induced with pulses of dim blue light (2.7 mW/cm2). These living, shape-changing materials may enable sensors or medical devices that respond to highly specific cues found within a biological milieu.


Assuntos
Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Resinas Acrílicas/farmacologia , Proliferação de Células/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Engenharia Genética , Optogenética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos
20.
Biochim Biophys Acta Mol Cell Res ; 1867(5): 118661, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31987792

RESUMO

Artemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detect cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells.


Assuntos
Artemisininas/farmacologia , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Artemisininas/química , Regulação para Baixo , Complexo III da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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