RESUMO
The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Glucose/metabolismo , Quinases da Glicogênio Sintase/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Aspergillus nidulans/enzimologia , Repressão Catabólica/genética , Fungos/genética , Fungos/metabolismo , Glicerol/metabolismo , Concentração Osmolar , Fosforilação/genética , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Xilose/metabolismoRESUMO
Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.
Assuntos
Aspergillus nidulans/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipídeos de Membrana/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62Awt) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7Å resolution model showed a five-bladed ß-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency.
Assuntos
Aspergillus nidulans/enzimologia , Celulose/farmacologia , Glicosídeo Hidrolases/química , Aspergillus nidulans/crescimento & desenvolvimento , Cristalografia , Glicosídeo Hidrolases/fisiologia , Glicosilação , Simulação de Dinâmica MolecularRESUMO
Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.
Assuntos
Aspergillus nidulans/enzimologia , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/química , Aspergillus nidulans/genética , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Especificidade por SubstratoRESUMO
Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/metabolismo , Plantas/química , Polissacarídeos/química , Aspergillus nidulans/genética , Biomassa , Proteínas Fúngicas/genética , Glucanos/química , Hidrólise , Pichia/genética , Pichia/metabolismo , Saccharum/química , Xilanos/químicaRESUMO
Este artigo propõe estudar os primeiros 12 anos de existência do Instituto de Radium de Minas Gerais, fundado em 1922. Sua atuação na luta contra o câncer no Brasil, ainda pouco conhecida, começa a ser esboçada pelo estudo de documentação institucional inédita. Através de um banco de dados elaborado com informações constantes em seu livro de registro de pacientes, foram feitos levantamentos estatísticos dos tipos de câncer e das formas de tratamento existentes entre 1923 e 1935. Esse livro faz parte de um conjunto de outros cinco recentemente descobertos no Centro de Memória da Medicina/UFMG. A documentação permite resgatar os primórdios das intervenções de radioterapia no país e acompanhar seu desenvolvimento e a influência exercida por esse hospital modelo.
This article proposes to study the first 12 years of the Minas Gerais Radium Institute, founded in 1922. Its work in the fight against cancer in Brazil, albeit still little known, is coming to light as its institutional documents are studied. A database has been prepared using information from its patient register, based on which statistical analyses have been done to identify the types of cancer and treatments available there between 1923 and 1935. This register is one of five recently unearthed at the Medicine Memory Center of the Universidade Federal de Minas Gerais. Through them, the earliest experiments in radiotherapy in Brazil can be reconstituted, and its development and the influence of this model hospital can be mapped out.
Assuntos
Feminino , Humanos , Masculino , Aspergillus nidulans/enzimologia , Dioxigenases , Ácido Homogentísico/análise , Oxigenases/metabolismo , Espectrofotometria/métodos , Alcaptonúria/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/metabolismo , Cromatografia Líquida de Alta Pressão , Ácido Homogentísico/metabolismo , Ácido Homogentísico/urina , Oxigenases/genética , Fenilacetatos/metabolismo , Fenilacetatos/farmacologia , Sensibilidade e EspecificidadeRESUMO
Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic stress, AtmA appears to perform a role in the regulation of TOR signaling, involving the retrograde and SnfA pathways. Thus, AtmA may represent a link between mitochondrial function and cell cycle or growth, possibly through the influence of the TOR and XprG function.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Autofagia , Carbono/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glioxilatos/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
In this article, the experiments used to construct the ambient pH-signaling network involved in the secretion of enzymes by filamentous fungi have been reviewed, focusing on the phosphate-repressible phosphatases in Aspergillus nidulans. Classic and molecular genetics have been used to demonstrate that proteolysis of the transcription factor PacC at alkaline ambient pH is imperative for its action, implying that the full-length version is not an active molecular form of PacC. It has been hypothesized that the transcriptional regulator PacC may be functional at both acidic and alkaline ambient pH, in either the full-length or the proteolyzed form, if it carries a pal-dependent molecular tag. The products of the pal genes are involved in a metabolic pathway that led to the synthesis of effector molecules that tag the pacC product, perhaps facilitating its proteolysis.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/fisiologia , Fosfatos/metabolismo , Fatores de Transcrição/fisiologia , Aspergillus nidulans/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Químicos , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Plant cell-wall arabinoxylans have a complex structure that requires the action of a pool of debranching (arabinofuranosidases) and depolymerizing enzymes (endo-xylanase). Two Aspergillus nidulans strains over-secreting endo-xylanase and arabinofuranosidase were inoculated in defined 2% maltose-minimum medium resulting in the simultaneously production of these enzymes. To study the synergistic hydrolysis was used arabinoxylan with 41% of arabinose and 59% of xylose residues. Thus, it was adopted different approaches to arabinoxylan hydrolysis using immobilized arabinofuranosidase and endo-xylanase: (i) endo-xylanase immobilized on glyoxyl agarose; (ii) arabinofuranosidase immobilized on glyoxyl agarose; (T1) hydrolysis of arabinoxylan with arabinofuranosidase immobilized on glyoxyl agarose for debranching, followed by a second hydrolysis with endo-xylanase immobilized on glyoxyl agarose; (T2) hydrolysis using (i) and (ii) simultaneously; and (T3) hydrolysis of arabinoxylan with endo-xylanase and arabinofuranosidase co-immobilized on glyoxyl agarose. It was concluded that arabinoxylan hydrolysis using two derivatives simultaneously (T2) showed greater hydrolytic efficiency and consequently a higher products yield. However, the hydrolysis with multi-enzymatic derivative (T3) results in direct release of xylose and arabinose from a complex substrate as arabinoxylan, which is a great advantage as biotechnological application of this derivative, especially regarding the application of biofuels, since these monosaccharides are readily assimilable for fermentation and ethanol production.
Assuntos
Aspergillus nidulans/enzimologia , Endo-1,4-beta-Xilanases/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Proteínas Imobilizadas/química , Xilanos/química , Arabinose/química , Aspergillus nidulans/química , Meios de Cultura , Endo-1,4-beta-Xilanases/isolamento & purificação , Fermentação , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Glioxilatos/química , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Imobilizadas/isolamento & purificação , Cinética , Sefarose/química , Especificidade por Substrato , Temperatura , Xilose/químicaRESUMO
Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/biossíntese , Expressão Gênica , Metaloendopeptidases/biossíntese , Pichia/genética , Sequência de Aminoácidos , Aspergillus nidulans/química , Aspergillus nidulans/genética , Clonagem Molecular , Meios de Cultura , Proteínas Fúngicas/genética , Metaloendopeptidases/genética , Metanol/metabolismo , Dados de Sequência Molecular , Pichia/metabolismoRESUMO
In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6-7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.
Assuntos
Aspergillus nidulans/enzimologia , Carbono/metabolismo , Divisão do Núcleo Celular , Proteínas Fúngicas/fisiologia , Fosfoinositídeo Fosfolipase C/fisiologia , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Glucose/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Upon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if the Aspergillus nidulans homologue of the EndoG gene, named nucA(EndoG), is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response in A. nidulans.
Assuntos
Aspergillus nidulans/enzimologia , Endodesoxirribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Apoptose/efeitos dos fármacos , Dano ao DNA , Endodesoxirribonucleases/genética , Farneseno Álcool/farmacologia , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/genética , Fenótipo , Proteínas Recombinantes de Fusão/genética , Regulação para CimaRESUMO
Previously, we demonstrated that the Aspergillus nidulans calC2 mutation in protein kinase C pkcA was able to confer tolerance to farnesol (FOH), an isoprenoid that has been shown to inhibit proliferation and induce apoptosis. Here, we investigate in more detail the role played by A. nidulans pkcA in FOH tolerance. We demonstrate that pkcA overexpression during FOH exposure causes increased cell death. FOH is also able to activate several markers of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Our results suggest an intense cross-talk between PkcA and the UPR during FOH-induced cell death. Furthermore, the overexpression of pkcA increases both mRNA accumulation and metacaspases activity, and there is a genetic interaction between PkcA and the caspase-like protein CasA. Mutant analyses imply that MAP kinases are involved in the signal transduction in response to the effects caused by FOH.
Assuntos
Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/enzimologia , Farneseno Álcool/farmacologia , Proteínas Fúngicas/metabolismo , Proteína Quinase C/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteína Quinase C/genéticaRESUMO
Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally, we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases, respectively, that have a function in FOH resistance. When exposed to FOH, the ΔaifA and ΔndeA strains have increased ROS production while ΔndeB, ΔndeA ΔndeB, and ΔndiA mutant strains showed the same ROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH.
Assuntos
Fator de Indução de Apoptose/metabolismo , Aspergillus nidulans/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Farneseno Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Mitocôndrias/enzimologia , Fator de Indução de Apoptose/genética , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Complexo I de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Mitocôndrias/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Pectinas/metabolismo , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Técnicas de Inativação de Genes , Proteína Quinase C/antagonistas & inibidores , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Aspergillus nidulans produces StcI esterase, which is involved in the biosynthesis of sterigmatocystin, a precursor of aflatoxins. Previous reports of this esterase in A. nidulans suggest that it is composed of 286 amino acid residues with a theoretical molecular mass of 31 kDa. Various conditions were evaluated to determine the optimal expression conditions for StcI; the highest level was observed when A. nidulans was cultured in solid oat media. Various esterases were expressed differentially according to the culture media used. However, specific antibodies designed to detect StcI reacted with a protein with an unexpected molecular mass of 35 kDa in cell extracts from all expression conditions. Analysis of the gene sequence and already reported expressed sequence tags indicated the presence of an additional 29-amino-acid N-terminal region of StcI, which is not a signal peptide and which has not been previously reported. We also detected the presence of this additional N-terminal region using reverse-transcriptase polymerase chain reaction. The complete protein (NStcI) was cloned and successfully expressed in Pichia pastoris.
Assuntos
Aspergillus nidulans/enzimologia , Esterases/genética , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Aspergillus nidulans/química , Aspergillus nidulans/genética , Sequência de Bases , Clonagem Molecular , Esterases/química , Esterases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/química , Fungos/classificação , Fungos/genética , Dados de Sequência Molecular , Peso Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here, we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/metabolismo , Oxirredutases/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/efeitos da radiação , Proteínas Fúngicas/genética , Deleção de Genes , Humanos , Peróxido de Hidrogênio/toxicidade , Metionina Sulfóxido Redutases , Viabilidade Microbiana , Oxidantes/toxicidade , Estresse Oxidativo , Oxirredutases/genética , Paraquat/toxicidade , Carbonilação Proteica , Raios UltravioletaRESUMO
Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The DeltaaifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 microM FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 microM FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.
Assuntos
Fator de Indução de Apoptose/genética , Aspergillus nidulans/genética , Farneseno Álcool/farmacologia , Proteínas Mitocondriais/genética , Oxirredutases/genética , Apoptose , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/enzimologia , Autofagia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Mitocôndrias/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Proteína Quinase C/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40 degrees C. The enzyme retained activity after incubation at pHs ranging from 8 to 11 for 12 h at 37 degrees C and 6 to 8 for 24 h at 37 degrees C. It retained 80% of its activity after incubation at 30 to 70 degrees C for 30 min and lost 50% of its activity after incubation for 15 min at 80 degrees C. Noticeable activation of the enzyme is observed when Fe(2+) ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu(2+), Fe(3+), Hg(2+), and Zn(2+) ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Endopeptidases/química , Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxilesterase/química , Carboxilesterase/genética , Carboxilesterase/isolamento & purificação , Carboxilesterase/metabolismo , Cátions/farmacologia , Endopeptidases/genética , Endopeptidases/metabolismo , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Compostos Orgânicos/análise , Análise de Sequência de Proteína , Especificidade por Substrato , TemperaturaRESUMO
The present study was designed to identify nutrient-dependent changes in extracellular pH and acid phosphatase secretion in the biA1 palC4 mutant strain of Aspergillus nidulans. The palC4 mutant was selected as lacking alkaline phosphatase, but having substantially increased acid phosphatase activity when grown on solid minimal medium under phosphate starvation, pH 6.5. Gene palC was identified as a putative member of a conserved signaling cascade involved in ambient alkaline sensing whose sole function is to promote the proteolytic activation of PacC at alkaline pH. We showed that both poor growth and conidiation of the palC4 mutant strain on solid medium, alkaline pH, were relative to its hypersensitivity to Tris (hydroxymethyl) aminomethane buffer. Also, the secretion of acid phosphatase was repressed when both the wild-type and palC4 mutant strains were grown in low-phosphate yeast extract liquid medium, pH 5.0, indicating that the secretion of this enzyme is not necessary to regenerate inorganic phosphate from the organic phosphate pool present in yeast extract.