The Experts below are selected from a list of 42 Experts worldwide ranked by ideXlab platform
Antonio J. Costa-filho - One of the best experts on this subject based on the ideXlab platform.
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Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension
Biochemical and Biophysical Research Communications, 2011Co-Authors: Sheila G. Couto, M. Cristina Nonato, Antonio J. Costa-filhoAbstract:Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a Flavine Mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme’s N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme’s active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.
Iuri Marques De Oliveira - One of the best experts on this subject based on the ideXlab platform.
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Nitroredutases : um estudo das possíveis funções na resposta ao estresse oxidativo
Universidade Federal do Rio Grande do Sul, 2013Co-Authors: Iuri Marques De OliveiraAbstract:As nitrorredutases compreendem uma família de enzimas dependentes de flavina adenina Mononucleotideo (FMN) capazes de metabolizar nitrocompostos usando nicotinamida adenina dinucleotídeo (NAD(P)H) como fonte de elétrons. Essas enzimas desempenham um papel central na metabolização de nitrocompostos recebendo grande atenção devido a sua habilidade em mediar a toxicidade desses compostos, tendo aplicações biotecnológicas e importância clínica. Essas enzimas podem ser encontradas em bactérias e em menor escala em fungos, protozoários e mamíferos. Em relação ao seu papel fisiológico, algumas hipóteses, como a participação na bioluminescência, homeostase metálica, biossíntese de cobalamina e resposta a estresse oxidativo têm sido propostas. Entretanto, não se tem conhecimento exato sobre a sua real função biológica. Neste cenário, este estudo tem como objetivo investigar possíveis funções das nitrorredutases no metabolismo de Escherichia coli e Saccharomyces cerevisiae, com ênfase na possível participação dessas enzimas na resposta ao estresse oxidativo. Para tanto, foi construída a rede de interações proteína-proteína das nitrorredutases NfsA e NfsB de E. coli e identificadas cinco sub-redes representando diferentes processos biológicos. Os resultados permitiram a elaboração de modelos sugerindo que as nitrorredutases de E. coli podem participar do metabolismo de ferro, manutenção do conteúdo de NADPH, metabolismo de compostos aromáticos e síntese de glicogênio. Estas vias podem contribuir nas respostas a estresse oxidativo e a limitação de nutrientes. Na levedura S. cerevisiae, foi determinada a influência das nitrorredutases Frm2p e Hbn1p na resposta a estresse oxidativo. Os resultados mostraram uma menor atividade basal de superóxido dismutase (SOD) e elevada sensibilidade a óxido de 4-nitroquinolina (4-NQO) e N-nitrosodietilamina (NDEA), indução de mutantes citoplasmáticos (petites), produção intracelular de ERO e peroxidação lipídica nas linhagens frm2 hbn1e frm2 hbn1quando expostas a estes agentes geradores de superóxido. Ainda foi observada elevada atividade basal de catalase (CAT), glutationa peroxidase (GPx) e conteúdo de glutationa (GSH) nas linhagens frm2 e frm2 hbn1. Estas linhagens possuem menor produção de espécies reativas de oxigênio (ERO) e peroxidação lipídica quando expostas aos peróxidos H2O2 e t-BOOH. Para elucidar os mecanismos pelos quais as nitrorredutases Frm2p e Hbn1p podem regular as defesas antioxidantes, foram identificadas, por biologia de sistemas, as interações dessas proteínas, tendo cinco sub-redes representando diferentes processos biológicos. Esta análise foi seguida por uma avaliação de índices de centralidade, que identificaram importantes proteínas da rede. Uma triagem dos fenótipos de sensibilidade a oxidantes foi realizada com linhagens proficientes e deficientes nestas proteínas identificadas e foram selecionadas as proteínas envolvidas nas respostas mais evidentes ao estresse oxidativo: as proteínas Ski8 (contribui na degradação do RNAm no sentido 3’-5’) e Cad1 (um ativador transcricional). Esta informação foi usada para a construção de linhagens duplo e triplo mutantes deficientes em Frm2p, Hbn1p, Ski8p ou Cad1p, seguindo a determinação da sensibilidade, acúmulo intracelular de ERO e nível de peroxidação lipídica na exposição a oxidantes e ainda a atividade basal de enzimas antioxidantes. Com base nos resultados obtidos, foi construído um modelo considerando que Cad1p ativa a expressão do gene FRM2 e a interação Frm2p-Ski8p regula as atividades das enzimas antioxidantes pela degradação do RNAm ou pela modulação da degradação dos transcritos do gene OLE1 (Ole1p atua na síntese de ácidos graxos insaturados) modificando a composição de ácidos graxos da membrana plasmática. A interação Hbn1p-Nab2p (Nab2p é necessária para a exportação do RNAm do núcleo para o citoplasma) controla a atividade de SOD pela exportação do RNAm.The nitroreductase family comprises a group of Flavine Mononucleotide (FMN)-dependent enzymes able to metabolize nitrosubstituted compounds using the reducing potential of nicotinamide adenine dinucleotide (NAD(P)H). Nitroreductase proteins play a central role in the activation of nitrocompounds and have received attention in recent decades based on their role in mediating nitrosubstituted compound toxicity, by its biotechnological application for bioremediation biocatalysis, and clinical importance in chemotherapeutic tumor treatment, ablation of specific cells and antibiotic resistance. Due to its relevance, different bacterial nitroreductases have been purified, and their biochemical, kinetic parameters and structure have been determined. Nitroreductases can be found within bacterial species and, in a less extend, in eukaryotes, such as fungi, protozoan and mammalian. A feature of the nitroreductase family is our lack of knowledge about its biological function. Therefore, new hypotheses have been proposed to solve the physiological role of nitroreductases, such as bioluminescence, metal homeostasis, vitamin B12 biosynthesis and oxidative stress response. In this context, this study aims to investigate possible functions of nitroreductases in Escherichia coli and Saccharomyces cerevisiae metabolism, with emphasis on possible role of these enzymes in oxidative stress response. Thus, a systems biology study was performed by generating protein-protein interactions (PPI) for NfsA and NfsB nitroreductases of E. coli. The results obtained from these systems biology analyses allow us to draw some models suggesting that E. coli nitroreductases can participate in iron metabolism, NADPH pool maintenance, aromatic compound metabolism, methionine and glycogen synthesis. In the yeast S. cerevisiae, the influence of Frm2p and Hbn1p nitroreductases in oxidative stress response was determined. The results showed a weaker basal activity of superoxide dismutase (SOD) and higher sensitivity for 4-nitroquinoline-oxide (4-NQO) and N-nitrosodiethylamine (NDEA), induction of petites, production of reactive oxygen species (ROS) and lipid peroxidation when exposed the these superoxide-generating agents. The results showed a higher basal activity of catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2 and frm2 hbn1. These strains were less ROS-producing and lipid peroxidation when exposed to peroxides-generating agents such as H2O2 and t-BOOH. To elucidate the mechanisms how nitroreductases regulate antioxidant defenses, we undertook a systems biology approach to identify Frm2p and Hbn1p interactions. A protein-protein interaction (PPI) network was obtained and biological processes related to RNA metabolism were observed. Thus, network centrality analysis was performed, which allows for selection of important proteins of network. A sensitivity screening of yeast strains proficient and deficient in these proteins to oxidants was performed and selected Ski8p (mediates 3'-5' RNA degradation) and Cad1p (transcriptional activator). This information was used to construct double and triple mutants defective for Frm2p, Hbn1p, Cad1p or Ski8p followed by determination of sensitivity, ROS accumulation, lipid peroxidation following oxidants exposure and basal antioxidant-enzyme activities. The results obtained allow us to draw model suggesting that Cad1p activate FRM2 following Frm2p-Ski8p interaction influences to oxidative stress response by regulates mRNA degradation of antioxidant-enzyme following their activities or OLE1 (Ole1p act in unsaturated fatty acid synthesis) transcripts degradation modifying the plasma membrane fatty acid composition. The Hbn1p-Nab2p (Nab2p act in mRNA export) interaction controls SOD activity by mRNA export
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Análise de duas possíveis nitrorredutases codificadas pelos genes FRM2 e HBN1 em Saccharomyces cerevisiae e suas funções na resposta ao estresse oxidativo
Universidade Federal do Rio Grande do Sul, 2008Co-Authors: Iuri Marques De OliveiraAbstract:As nitrorredutases compreendem uma família de proteínas conservadas evolutivamente e originalmente identificadas em eubactérias. São enzimas capazes de catalisar a redução do grupo nitro e utilizam FMN (flavina Mononucleotideo) ou FAD (flavina adenina dinucleotídeo oxidado) como grupo prostético e NADPH (nicotinamida adenina dinucleotídeo fosfato reduzido) ou NADH (nicotinamida adenina dinucleotídeo reduzido) como agentes redutores. As nitrorredutases podem ser encontradas em bactérias e em menor extensão em eucariotos. Dois subgrupos de nitrorredutases foram caracterizados em bactérias: oxigênio-insensível ou tipo I e oxigênio-sensível ou tipo II. Na levedura Saccharomyces cerevisiae duas prováveis nitrorredutases, Frm2p/Hbn1p foram identificadas. Em relação às enzimas pertencentes à família das nitrorredutases não se tem conhecimento sobre a sua função biológica, bem como em relação à sua posição filogenética. Tendo isso em vista, o objetivo deste trabalho é esclarecer a possível função das proteínas Frm2 e Hbn1 de Saccharomyces cerevisiae na resposta ao estresse oxidativo, bem como determinar a posição filogenética e a sua presença em outros organismos procariotos e eucariotos. Os resultados da análise filogenética mostram que bactérias possuem seqüências similares a Frm2p/Hbn1p (denominadas Nr1Ap) que formam um clado distinto dentro da família Frm2p/Hbn1p. Análises de agrupamentos hidrofóbicos (HCA) e modelagem tri-dimensional foram realizadas para comparar regiões conservadas entre proteínas Nr1Ap e Frm2p/Hbn1p. A nitrorredutase Frm2p possivelmente esteja atuando na via de sinalização lipídica, enquanto a função da Hbn1p é desconhecida. Entretanto, alguns estudos têm indicado que as nitrorredutases podem estar envolvidas na resposta a estresse oxidativo. Com o objetivo de esclarecer a função de Frm2p e Hbn1p, foi avaliada a sensibilidade de linhagens de levedura proficientes e deficientes em ambas proteínas ao estresse oxidativo, investigando a competência respiratória, as atividades de enzimas antioxidantes, a produção intracelular de espécies reativas de oxigênio (EROs) e a peroxidação lipídica. Os resultados mostram uma menor atividade basal de superóxido dismutase (SOD) e elevada sensibilidade a óxido de 4-nitroquinolina (4-NQO) e Nnitrosodietilamina (NDEA), indução de mutantes citoplasmáticos (petites), produção intracelular de EROs e peroxidação lipídica quando expostas a estes agentes geradores de superóxido nas linhagens frm2 , hbn1 e frm2 hbn1 . Ainda podemos observar elevada atividade basal de catalase (CAT), glutationa peroxidase (GPx) e conteúdo de glutationa (GSH) nas linhagens frm2 e frm2 hbn1. Estas linhagens possuem menor produção de EROs e peroxidação lipídica quando expostas aos agentes geradores de peróxidos H2O2 e t-BOOH. Isso sugere que a ausência da Frm2p é o fator responsável por estas alterações vistas. Portanto, neste trabalho foi mostrada a influência das nitrorredutases Frm2 e Hbn1 na resposta ao estresse oxidativo em S. cerevisiae, pela modulação da atividade das enzimas antioxidantes, SOD, CAT e GPx, bem como do conteúdo de GSH. Adicionalmente também foi constatado que as nitrorredutases Frm2p e Hbn1p provavelmente não atuam na metabolização de nitrocompostos. Estes resultados são consistentes com os dados encontrados na análise filogenética, que apontam estas proteínas como constituindo uma nova família de prováveis nitrorredutases ainda não caracterizada, encontrada em bactérias e fungos.The nitroreductase family comprises a group of FMN (Flavine Mononucleotide) ou FAD (Flavine adenine dinucleotíde oxidade)-dependent enzymes able to metabolize nitrosubstituted compounds using the reducing power of NADPH (nicotinamide adenine dinucleotide fosfate reduzide) or NADH (nicotinamide adenine dinucleotide reduzide). The nitroreductases can be found within bacterial species and, in a less extend, in eukaryotes. Two types of nitroreductase subgroups were characterized in bacteria: oxygen-insensitive or type I and oxygen sensitive or type II. In the yeast Saccharomyces cerevisiae two putative nitroreductase proteins, Frm2p and Hbn1p, were described. A feature of the nitroreductase family is our lack of knowledge about its biological function and evolutionary history. Taking into account these considerations, the purpose of this work was to elucidate the possible participation of these enzymes in response to oxidative stress as well as to determine the phylogenetic position of Frm2p and Hbn1p and the presence of homologous sequences in other prokaryotic and eukaryotic species. In order to obtain data about the phylogenetic position of Frm2p/Hbn1p, we performed an in-depth phylogenetic analysis of these proteins. The phylogenetic analysis of these proteins showed that bacterial cells have a Frm2p/Hbn1p-like sequences (termed NrlAp) which form a distinct clade within the fungal Frm2p/Hbn1p family. Hydrophobic cluster analysis (HCA) and three-dimensional protein modeling allowed us to compare conserved regions among NrlAp and Frm2/Hbn1p proteins. While Frm2p appears to act in the lipid signaling pathway, the function of Hbn1p is unknown. However, some works suggests a possible involvement from the nitroreductases in response the stress oxidative. In order to elucidate the functions of Frm2p and Hbn1p, we evaluate the sensitivity of proficient and deficient yeast strains for both proteins for oxidative stress, considering the respiratory competence, antioxidant enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. The results showed a weaker basal activity of superoxide dismutase (SOD) and higher sensitivity for 4-nitroquinoline-oxide (4-NQO) and NNitrosodiethylamine (NDEA), induction of petites, ROS production and lipid peroxidation when exposed the these superoxide generating agents. The results showed a higher basal activity of catalase (CAT), glutathione peroxidase (Gpx) and reduced glutathione (GSH) content in the single and double mutant strains frm2 and frm2 hbn1 . These strains were less ROS-producing and lipid peroxidation when exposed to peroxides-generating agents H2O2 and t-BOOH. Thus, the absence of Frm2p may be the event responsible for these alterations. Considering the data gathered in this work, we showed the influence of nitroreductases Frm2p and Hbn1p in response to oxidative stress in S. cerevisae yeast by modulation of antioxidant enzymes activities, such as SOD, CAT, GPx and GSH content. Additionally, it was showed that the nitroreductases Frm2p and Hbn1p are not envolved in the activation of nitrocompounds. Theses results are consistent with those found in the filogenetic analysis that indicated that theses proteins belong to the new bacterial and fungal Frm2p/Hbn1p nitroreductase-like family
Sheila G. Couto - One of the best experts on this subject based on the ideXlab platform.
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Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension
Biochemical and Biophysical Research Communications, 2011Co-Authors: Sheila G. Couto, M. Cristina Nonato, Antonio J. Costa-filhoAbstract:Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a Flavine Mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme’s N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme’s active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.
M. Cristina Nonato - One of the best experts on this subject based on the ideXlab platform.
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Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension
Biochemical and Biophysical Research Communications, 2011Co-Authors: Sheila G. Couto, M. Cristina Nonato, Antonio J. Costa-filhoAbstract:Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a Flavine Mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme’s N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme’s active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.
Vogel R.o. - One of the best experts on this subject based on the ideXlab platform.
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The assembly of mitochondrial Complex I. A product of nuclear-mitochondrial synergy.
[S.l. : s.n.], 2007Co-Authors: Vogel R.o.Abstract:Mitochondria are essential to cellular energy production. Embedded in the mitochondrial inner membrane, the engine of the mitochondrial powerhouse is formed by the five enzymatic complexes of the oxidative phosphorylation (OXPHOS) system. Dysfunction of this system results in mitochondrial disease, affecting organs such as brain, eyes, heart, muscle, liver and kidneys, often resulting in early childhood death. Most frequently affected is the largest multi-protein complex of the OXPHOS system, termed complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Its assembly is a formidable cellular achievement. The process encompasses the combination of 38 nuclear DNA-encoded and seven mitochondrial DNA-encoded constituents, eight iron-sulfur clusters and a noncovalently bound Flavine Mononucleotide, resulting in one of the most complex structures in the mitochondrion. This thesis describes studies which demonstrate key stages in the assembly process and which identify and characterize chaperone proteins which aid the process. This has led to a detailed model for complex I assembly and the identification of a new chaperone protein, Ecsit. Ecsit was previously known from its role in the immune system. Its mitochondrial function in the assembly of complex I provides yet another link between the assembly of OXPHOS complexes and cellular processes, such as previously demonstrated for regulated cell death (apoptosis) and fatty acid synthesis. Hopefully, these insights will contribute to the understanding of why mitochondrial dysfunction is so important in e.g. diabetes, Alzheimer's and Parkinson's disease, ageing and cancer
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The assembly of mitochondrial Complex I. A product of nuclear-mitochondrial synergy.
[S.l. : s.n.], 2007Co-Authors: Vogel R.o.Abstract:Contains fulltext : 30935_asseofmic.pdf (publisher's version ) (Open Access)Mitochondria are essential to cellular energy production. Embedded in the mitochondrial inner membrane, the engine of the mitochondrial powerhouse is formed by the five enzymatic complexes of the oxidative phosphorylation (OXPHOS) system. Dysfunction of this system results in mitochondrial disease, affecting organs such as brain, eyes, heart, muscle, liver and kidneys, often resulting in early childhood death. Most frequently affected is the largest multi-protein complex of the OXPHOS system, termed complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Its assembly is a formidable cellular achievement. The process encompasses the combination of 38 nuclear DNA-encoded and seven mitochondrial DNA-encoded constituents, eight iron-sulfur clusters and a noncovalently bound Flavine Mononucleotide, resulting in one of the most complex structures in the mitochondrion. This thesis describes studies which demonstrate key stages in the assembly process and which identify and characterize chaperone proteins which aid the process. This has led to a detailed model for complex I assembly and the identification of a new chaperone protein, Ecsit. Ecsit was previously known from its role in the immune system. Its mitochondrial function in the assembly of complex I provides yet another link between the assembly of OXPHOS complexes and cellular processes, such as previously demonstrated for regulated cell death (apoptosis) and fatty acid synthesis. Hopefully, these insights will contribute to the understanding of why mitochondrial dysfunction is so important in e.g. diabetes, Alzheimer's and Parkinson's disease, ageing and cancer.RU Radboud Universiteit Nijmegen, 14 november 2007Promotor : Smeitink, J.A.M. Co-promotor : Nijtmans, L.G.J.264 p