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Luis E S Netto

Publications and source records attributed to Luis E S Netto.

9 recordsLinked to original sources
PubMed2006

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging.

Replicative life span in Saccharomyces cerevisiae is increased by glucose (Glc) limitation [calorie restriction (CR)] and by augmented NAD+. Increased survival promoted by CR was attributed previously to the NAD+-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD+ synthesis and salvage pathways (pnc1delta, npt1delta, and bna6delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1delta and bna6delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

Base Sequence↗
PubMed2006

The structural molecular biology network of the State of São Paulo, Brazil.

This article describes the achievements of the Structural Molecular Biology Network (SMolBNet), a collaborative program of structural molecular biology, centered in the State of São Paulo, Brazil, and supported by São Paulo State Funding Agency (FAPESP). It gathers twenty scientific groups and is coordinated by the scientific staff of the Center of Structural Molecular Biology, at the National Laboratory of Synchrotron Light (LNLS), in Campinas. The SMolBNet program has been aimed at 1) solving the structure of proteins of interest related to the research projects of the groups. In some cases, the choice has been to select proteins of unknown function or of possible novel structure obtained from the sequenced genomes of the FAPESP genomic program; 2) providing the groups with training in all the steps of the protein structure determination: gene cloning, protein expression, protein purification, protein crystallization and structure determination. Having begun in 2001, the program has been successful in both aims. Here, four groups reveal their participation in the program and describe the structural aspects of the proteins they have selected to study.

Brazil↗
PubMed2006

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.

Animals↗
PubMed2006

Yeast oxidative stress response. Influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state.

We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability. First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death. This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2. We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense. On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2-mediated activation of some genes otherwise observed, impairing cell defense and viability. The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder. Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation/deactivation. Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression.

Cytoplasm↗
PubMed2005

Heat stress promotes mitochondrial instability and oxidative responses in yeast deficient in thiazole biosynthesis.

The Thi4 protein from Saccharomyces cerevisiae plays a pivotal role in the biosynthesis of thiazole, a precursor of thiamine (vitamin B1). In addition, the thi4-disrupted strain has shown increased frequencies of mitochondrial mutants (petite colonies) upon treatment with DNA damaging agents. In this work, we show that the thi4 strain presents significant induction of petites and reduced oxygen consumption when grown at 37 degrees C, a condition that induces high levels of reactive oxygen species in yeast. Oxidative stress parameters were thus measured in thi4 cells. The activities of superoxide dismutase and phospholipid hydroperoxide glutathione peroxidase were significantly increased when these mutants were grown at 37 degrees C compared to the wild-type strain (W303). The levels of carbonyl protein groups were also significantly higher for the thi4 strain than for W303. Still, significant reductions in protein thiols and reduced glutathione were observed for the mutated strain. Therefore, the Thi4 protein appears to play an important protective role during heat stress in yeast cells, a feature probably related to the mitochondrial instability and altered oxidative status observed in thi4 mutants.

Glutathione↗
PubMed2004

Glutathione and thioredoxin peroxidases mediate susceptibility of yeast mitochondria to Ca(2+)-induced damage.

The effect of thioredoxin peroxidases on the protection of Ca(2+)-induced inner mitochondrial membrane permeabilization was studied in the yeast Saccharomyces cerevisiae using null mutants for these genes. Since deletion of a gene can promote several other effects besides the absence of the respective protein, characterizations of the redox state of the mutant strains were performed. Whole cellular extracts from all the mutants presented lower capacity to decompose H(2)O(2) and lower GSH/GSSG ratios, as expected for strains deficient for peroxide-removing enzymes. Interestingly, when glutathione contents in mitochondrial pools were analyzed, all mutants presented lower GSH/GSSG ratios than wild-type cells, with the exception of DeltacTPxI strain (cells in which cytosolic thioredoxin peroxidase I gene was disrupted) that presented higher GSH/GSSG ratio. Low GSH/GSSG ratios in mitochondria increased the susceptibility of yeast to damage induced by Ca(2+) as determined by membrane potential and oxygen consumption experiments. However, H(2)O(2) removal activity appears also to be important for mitochondria protection against permeabilization because exogenously added catalase strongly inhibited loss of mitochondrial potential. Moreover, exogenously added recombinant peroxiredoxins prevented inner mitochondrial membrane permeabilization. GSH/GSSG ratios decreased after Ca(2+) addition, suggesting that reactive oxygen species (ROS) probably mediate this process. Taken together our results indicate that both mitochondrial glutathione pools and peroxide-removing enzymes are key components for the protection of yeast mitochondria against Ca(2+)-induced damage.

Calcium↗
PubMed2003

H(2)O(2) generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c.

Impaired electron transport chain function has been related to increases in reactive oxygen species (ROS) generation. Here we analyzed different pet mutants of Saccharomyces cerevisiae in order to determine the relative contribution of respiratory chain components in ROS generation and removal. We found that the maintenance of respiration strongly prevented mitochondrial H(2)O(2) release and increased cellular H(2)O(2) removal. Among all respiratory-deficient strains analyzed, cells lacking cytochrome c (cyc3 point mutants) presented the highest level of H(2)O(2) synthesis, indicating that the absence of functional cytochrome c in mitochondria leads to oxidative stress. This finding was supported by the presence of high levels of catalase and peroxidase activity despite the lack of respiration. Furthermore, the addition of exogenous cytochrome c to isolated yeast mitoplasts significantly reduced H(2)O(2) detection in a manner enhanced by cytochrome c reduction and the presence of a functional respiratory chain. Together, our results indicate that the maintenance of electron transport by cytochrome c prevents ROS generation by the respiratory chain.

Animals↗