RESUMO
Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.
Assuntos
Álcool Desidrogenase , Proteínas de Arabidopsis , Arabidopsis , Oxirredução , Arabidopsis/enzimologia , Arabidopsis/genética , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Especificidade por Substrato , S-Nitrosoglutationa/metabolismo , Sequência de Aminoácidos , Etanol/metabolismoRESUMO
Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).
RESUMO
Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified A. thaliana AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in hot5-2/gsnor1-3 mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.
RESUMO
Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRXIIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell.
RESUMO
Recent studies suggest cysteine S-nitrosation of S-nitrosoglutathione reductase (GSNOR) could regulate protein redox homeostasis. "Switch" assays enable discovery of putatively S-nitrosated proteins. However, with few exceptions, researchers have not examined the kinetics and biophysical consequences of S-nitrosation. Methods to quantify protein S-nitrosothiol (SNO) abundance and formation kinetics would bridge this mechanistic gap and allow interpretation of the consequences of specific modifications, as well as facilitate development of specific S-nitrosation inhibitors. Here, we describe a rapid assay to estimate protein SNO abundance with intact protein electrospray ionization mass spectrometry. Originally designed using recombinant GSNOR, these methods are applicable to any purified protein to test for or further study nitrosatable cysteines.
Assuntos
Aldeído Oxirredutases/análise , S-Nitrosotióis/análise , Espectrometria de Massas por Ionização por Electrospray , Nitrosação , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Reactive molecular species (RMS) can damage DNA, lipids, and proteins but as signaling molecules they also affect the regulatory state of the cell. RMS consist of reactive oxygen (ROS), nitrogen (RNS), and carbonyl species (RCS). Besides their potentially destructive nature, RMS are able to modify proteins at the posttranslational level, resulting in regulation of structure, activity, interaction as well as localization. This chapter addresses methods to analyze and quantify posttranslational redox modifications in vitro and ex vivo, such as sulfenic acid generation of cysteine residues and oxidative carbonylation of proteins. In addition, by use of isothermal titration calorimetry, redox-dependent interaction studies of proteins will be described.
Assuntos
Carbonilação Proteica , Processamento de Proteína Pós-Traducional , Varredura Diferencial de Calorimetria/métodos , OxirreduçãoRESUMO
Redox homeostasis is an important parameter of cell function and cell signaling. Spatial and temporal alterations of redox state control metabolism, developmental processes, as well as acute responses to environmental stresses and stress acclimation. Redox homeostasis is also linked to the circadian clock. This chapter introduces methods to assess important redox parameters such as the low molecular weight redox metabolites glutathione and ascorbate, their amount and redox state, and H2O2 as reactive oxygen species. In vivo redox cell imaging is described by use of the reduction-oxidation sensitive green fluorescent protein (roGFP). Finally, on the level of posttranslational redox modifications of proteins, methods are shown to assess hyperoxidation of 2-cysteine peroxiredoxin and glutathionylation of peroxiredoxin IIE. The redox state of 2-cysteine peroxiredoxin has been identified as a transcription-independent marker of circadian rhythmicity.
Assuntos
Ritmo Circadiano/fisiologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Cisteína/metabolismo , Expressão Gênica , Genes Reporter , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismoRESUMO
BACKGROUND: Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown. METHODS: The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential. RESULTS: Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. CONCLUSIONS: The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.