RESUMO
Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , NADP/metabolismo , Dióxido de Carbono/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Cloroplastos/metabolismo , Oxirredução , Folhas de Planta/metabolismo , Tiorredoxinas/metabolismo , AclimataçãoRESUMO
Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/ß-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.
RESUMO
Nucleoredoxin (NXN), an oxidoreductase enzyme, contributes to cellular redox homeostasis by regulating different signaling pathways in a redox-dependent manner. By interacting with seven proteins so far, namely disheveled (DVL), protein phosphatase 2A (PP2A), phosphofructokinase-1 (PFK1), translocation protein SEC63 homolog (SEC63), myeloid differentiation primary response gene-88 (MYD88), flightless-I (FLII), and calcium/calmodulin-dependent protein kinase II type alpha (CAMK2A), NXN is involved in the regulation of several key cellular processes, including proliferation, organogenesis, cell cycle progression, glycolysis, innate immunity and inflammation, motility, contraction, protein transport into the endoplasmic reticulum, neuronal plasticity, among others; as a result, NXN has been implicated in different pathologies, such as cancer, alcoholic and polycystic liver disease, liver fibrogenesis, obesity, Robinow syndrome, diabetes mellitus, Alzheimer's disease, and retinitis pigmentosa. Together, this evidence places NXN as a strong candidate to be a master redox regulator of cell physiology and as the hub of different redox-sensitive signaling pathways and associated pathologies. This review summarizes and discusses the current insights on NXN-dependent redox regulation and its implication in different pathologies.
RESUMO
KEY MESSAGE: This review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.
Assuntos
Evolução Molecular , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/fisiologia , Plantas/enzimologia , Regulação Alostérica , Glucose-1-Fosfato Adenililtransferase/genética , Modelos Moleculares , Conformação Proteica , Amido/biossíntese , Amido/químicaRESUMO
Glycine betaine (N, N, N-trimethyl amine) is an osmolyte accumulated in cells that is key for cell volume and turgor regulation, is the principal methyl donor in the methionine cycle and is a DNA and proteins stabilizer. In humans, glycine betaine is synthesized from choline and can be obtained from some foods. Glycine betaine (GB) roles are illustrated in chemical, metabolic, agriculture, and clinical medical studies due to its chemical and physiological properties. Several studies have extensively described GB role and accumulation related to specific pathologies, focusing mainly on analyzing its positive and negative role in these pathologies. However, it is necessary to explain the relationship between glycine betaine and different pathologies concerning its role as an antioxidant, ability to methylate DNA, interact with transcription factors and cell receptors, and participate in the control of homocysteine concentration in liver, kidney and brain. This review summarizes the most important findings and integrates GB role in neurodegenerative, cardiovascular, hepatic, and renal diseases. Furthermore, we discuss GB impact on other dysfunctions as inflammation, oxidative stress, and glucose metabolism, to understand their cross-talks and provide reliable data to establish a base for further investigations.
Assuntos
Betaína/metabolismo , Doenças Cardiovasculares/metabolismo , Nefropatias/metabolismo , Hepatopatias/metabolismo , Doenças Neurodegenerativas/metabolismo , Tamanho Celular , Humanos , Hiper-Homocisteinemia/metabolismo , Concentração Osmolar , S-Adenosilmetionina/metabolismoRESUMO
Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.
Assuntos
Glutarredoxinas , Glutationa , Catálise , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , FilogeniaRESUMO
Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O2 conditions. However, an increase in the stoichiometry of photorespiratory CO2 release was found during O2 -dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD+ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Tiorredoxina h/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dióxido de Carbono/metabolismo , Respiração Celular/fisiologia , Clorofila A , Regulação da Expressão Gênica de Plantas , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina Hidroximetiltransferase , Oxirredução , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Tiorredoxina h/genética , TranscriptomaRESUMO
In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.
Assuntos
Cisteína/genética , Longevidade , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina/genética , Glutationa/metabolismo , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Redox regulation in biology is largely operated by cysteine chemistry in response to a variety of cell environmental and intracellular stimuli. The high chemical reactivity of cysteines determines their conservation in functional roles, but their presence can also result in harmful oxidation limiting their general use by proteins. Papillomaviruses constitute a unique system for studying protein sequence evolution since there are hundreds of anciently evolved stable genomes. E7, the viral transforming factor, is a dimeric, cysteine-rich oncoprotein that shows both conserved structural and variable regulatory cysteines constituting an excellent model for uncovering the mechanism that drives the acquisition of redox-sensitive groups. By analyzing over 300 E7 sequences, we found that although noncanonical cysteines show no obvious sequence conservation pattern, they are nonrandomly distributed based on topological constrains. Regulatory residues are strictly excluded from six positions stabilizing the hydrophobic core while they are enriched in key positions located at the dimerization interface or around the Zn+2 ion. Oxidation of regulatory cysteines is linked to dimer dissociation, acting as a reversible redox-sensing mechanism that triggers a conformational switch. Based on comparative sequence analysis, molecular dynamics simulations and biophysical analysis, we propose a model in which the occurrence of cysteine-rich positions is dictated by topological constrains, providing an explanation to why a degenerate pattern of cysteines can be achieved in a family of homologs. Thus, topological principles should enable the possibility to identify hidden regulatory cysteines that are not accurately detected using sequence based methodology.
Assuntos
Cisteína , Evolução Molecular , Proteínas E7 de Papillomavirus/genética , Sequência de Aminoácidos , DimerizaçãoRESUMO
In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the alpha5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (alpha5-C76S or alpha5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that alpha5-C76S or alpha5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random alpha5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., a-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the alpha5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the a5-subunit, and consequently impacts the lifespan of yeast.
RESUMO
In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (ß-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.
RESUMO
Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Fumarato Hidratase/química , Fumaratos/química , Regulação da Expressão Gênica de Plantas , Malato Desidrogenase/química , Regulação Alostérica , Arabidopsis/química , Arabidopsis/classificação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Asparagina/metabolismo , Sítios de Ligação , Evolução Molecular , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Expressão Gênica , Glutamina/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Modelos Moleculares , NADP/metabolismo , Ácido Oxaloacético/metabolismo , Oxirredução , Filogenia , Agregados Proteicos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ΔphaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ΔphaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.
RESUMO
Thyroid hormone receptors (TRs) are responsible for mediating thyroid hormone (T3 and T4) actions at a cellular level. They belong to the nuclear receptor (NR) superfamily and execute their main functions inside the cell nuclei as hormone-regulated transcription factors. These receptors also exhibit so-called "non-classic" actions, for which other cellular proteins, apart from coregulators inside nuclei, regulate their activity. Aiming to find alternative pathways of TR modulation, we searched for interacting proteins and found that PDIA1 interacts with TRß in a yeast two-hybrid screening assay. The functional implications of PDIA1-TR interactions are still unclear; however, our co-immunoprecipitation (co-IP) and fluorescence assay results showed that PDI was able to bind both TR isoforms in vitro. Moreover, T3 appears to have no important role in these interactions in cellular assays, where PDIA1 was able to regulate transcription of TRα and TRß-mediated genes in different ways depending on the promoter region and on the TR isoform involved. Although PDIA1 appears to act as a coregulator, it binds to a TR surface that does not interfere with coactivator binding. However, the TR:PDIA1 complex affinity and activation are different depending on the TR isoform. Such differences may reflect the structural organization of the PDIA1:TR complex, as shown by models depicting an interaction interface with exposed cysteines from both proteins, suggesting that PDIA1 might modulate TR by its thiol reductase/isomerase activity.
RESUMO
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Assuntos
Aldeído Redutase/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Prunus domestica/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Frutosedifosfatos/metabolismo , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Dissulfeto de Glutationa/metabolismo , Hexosefosfatos/metabolismo , Hexosefosfatos/farmacologia , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismoRESUMO
In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (ß-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.
RESUMO
The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.
Assuntos
Giardia lamblia/enzimologia , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/isolamento & purificação , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Giardia lamblia/genética , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Oxirredução , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Glutathione peroxidases are thiol-based enzymes that catalyze the reduction of H2O2 and hydroperoxides to H2O or alcohols, they mitigate the toxicity of these compounds to the cell mainly using thioredoxin as an electron donor. Additionally, certain redox sensor and signaling functions are being ascribed to these enzymes in prokaryotes, fungi, and plants. We review the evolutionary history, enzymatic and biochemical evidence that make GPX proteins, in addition to being peroxiredoxins, important candidates for acting as redox sensor proteins in plants: (i) the lower peroxidase activity of Cys-GPX; (ii) the thiol catalytic center; (iii) the capacity to interact with regulatory proteins. All these characteristics suggest that at the basal level, plant GPXs have an important role in redox signal transduction in addition to their peroxidase activity.