Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

Cadmium induces senescence symptoms in leaf peroxisomes of pea plants

The effect of growing pea (Pisum sativum L.) plants with a toxic CdCl₂ concentration (50 µm ) on the metabolism and proteolytic activity of leaf peroxisomes was studied. In peroxisomes purified from plants treated with cadmium, an increase in the total protein concentration and in the activity and protein level of the photorespiratory enzyme glycolate oxidase was found. The glyoxylate cycle enzymes, malate synthase and isocitrate lyase, whose activity is normally very low in leaf peroxisomes, were enhanced by Cd treatment. The activity of the endogenous proteases of leaf peroxisomes was determined. Two leucine‐aminopeptidase isozymes (AP1‐AP2) were detected, and their activity was slightly higher in Cd‐treated plants. Five endopeptidases (EP1‐EP5) were present in pea leaf peroxisomes, and in plants grown with Cd the activity of isozymes EP1‐EP4 was increased. The ultrastructural analysis of pea leaves showed that Cd produced a disorganization of the chloroplast structure, with an increase in the number of plastoglobuli, and the formation of vesicles in the vacuoles. Taken together, these results indicate that Cd induces senescence symptoms in leaf peroxisomes, and probably a metabolic transition of leaf peroxisomes into glyoxysomes, and suggest that the peroxisomal proteases could participate in the metabolic changes produced by Cd.     

Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria

The effect in vivo of salt stress on the activated oxygen metabolism of mitochondria, was studied in leaves from two NaCl-treated cultivars of Pisum sativum L. with different sensitivity to NaCl. In mitochondria from NaCl-sensitive plants, salinity brought about a significant decrease of Mn-SOD (EC 1. 15. 1. 1) Cu, Zn-SOD I (EC 1. 15. 1. 1) and fumarase (EC 4. 2. 1. 2) activities. Conversely, in salt-tolerant plants NaCl treatment produced an increase in the mitochondrial Mn-SOD activity and, to a lesser extent, in fumarase activity. In mitochondria from both salt-treated cultivars, the internal H₂O₂ concentration remained unchanged. The NADH- and succinate-dependent generation of O₂^.−radicals by submitochondrial particles and the lipid peroxidation of mitochondrial membranes, increased as a result of salt treatment, and these changes were higher in NaCl-sensitive than in NaCl-tolerant plants. Accordingly, the enhanced rates of superoxide production by mitochondria from salt-sensitive plants were concomitant with a strong decrease in the mitochondrial Mn-SOD activity, whereas NaCl-tolerant plants appear to have a protection mechanism against salt-induced increased O₂^.− production by means of the induction of the mitochondrial Mn-SOD activity. These results indicate that in the subcellular toxicity of NaCl in pea plants, at the level of mitochondria, an oxidative stress mechanism mediated by superoxide radicals is involved, and also imply a function for mitochondrial Mn-SOD in the molecular mechanisms of plant tolerance to NaCl.     

S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: changes under abiotic stress

Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.     

Analysis of peroxiredoxin decreasing oxidative stress in hypertensive aortic smooth muscle

To determine the role of peroxiredoxin (Prx) in response to oxidative stress and during hypertension in the vasculature, we identified Prx proteins and analyzed their antioxidant effects. Rat aortic smooth muscle contains all six Prxs (I-VI). Prx I, II, and VI shifted to its acidic site on two-dimensional polyacrylamide gel electrophoresis after exposure to H(2)O(2). The total expression of Prx I and VI was increased in response to H(2)O(2). The expression of Prx I, but not that of Prx II and VI, increases and the acidic form of Prx I and the sulfonic acid form of Prx (SO(3)H-Prx) are more strongly expressed in the aortic smooth muscle of hypertensive rats than in that of normotensive control rats. Prxs were also found in the mesenteric artery, heart, and kidney. The expression levels of Prx I and VI were increased in mesenteric artery, but not heart and kidney, from hypertensive rats compared with that from normotensive rats. These results suggest that Prxs play a crucial role against oxidative stress in vascular smooth muscles during hypertension.     

Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.     

Contribution of mitochondria and peroxisomes to palmitate oxidation in pea tissues

Total, mitochondrial and peroxisomal palmitate oxidation capacities were compared in pea, from the dry seed to 14 days after imbibition. Total beta-oxidation varied over the measured time period and showed four peaks of activity at day 2, days 5-6, day 10 and days 12-13. The contribution of peroxisomal and mitochondrial beta-oxidation to this overall beta-oxidation varied. Over the first 48 h of seed germination, peroxisomal beta-oxidation accounted for 80-100% of the total observed beta-oxidation. The larger peaks of beta-oxidation at days 5-6, day 10 and days 12-13 were due primarily to mitochondrial beta-oxidation activity, which accounted for 70-90% of the observed total beta-oxidation at these times. The peaks of activity are related to observed stages in seedling development.     

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

ATM activation in the presence of oxidative stress

The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H₂O₂. The active ATM in this case is a disulfide-crosslinked dimer containing two or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSB s is inhibited in the presence of H₂O₂ because oxidation blocks the ability of MRN to bind to DNA . However, ATM activation via direct oxidation by H₂O₂ complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSB s and oxidative stress.Key words: ATM, DNA repair, double-strand break, oxidative stress, ROS     

Effect of sublethal 6-hydroxydopamine on the response to subsequent oxidative stress in dopaminergic cells: evidence for preconditioning

Exposure to sublethal stress can trigger endogenous protection against subsequent, higher levels of stress. We tested for this preconditioning phenomenon in a model of Parkinson's disease by applying 6-hydroxydopamine to the dopaminergic MN9D cell line. Exposure to sublethal concentrations of 6-hydroxydopamine (5-10 microM) protected against the toxic effects of a subsequent exposure to a higher concentration (50 microM), as measured by the Hoechst assay for nuclear viability. This was accompanied by little or no protection against 6-hydroxydopamine-induced lactate dehydrogenase release, decline in ATP, or reduction in (3)H-dopamine uptake. The antioxidant, N-acetyl cysteine (20 mM), when applied during preconditioning, abolished protection, as did the protein synthesis inhibitor, cycloheximide (0.2 microM). Preconditioning did not affect superoxide dismutase or glutathione peroxidase enzymes, or levels of heat shock protein-72. However, Bcl-2 protein levels rose with preconditioning. Preconditioning rapidly increased phosphorylation of kinases ERK1/2, Akt and JNK, and was abolished by pharmacological inhibitors of their activity. Finally, sublethal 6-hydroxydopamine preconditioned against the toxicity of proteasome inhibitor, MG-132 (1 microM). Thus, exposure of a dopaminergic cell line to sublethal oxidative stress can protect against additional oxidative stress due to translational and post-translational modifications, as well as confer 'cross-tolerance' against a different insult, proteasome inhibition.     

Immunological evidence for the presence of ribulose bisphosphate carboxylase in guard cell chloroplasts

Ribulose bisphosphate carboxylase (Rubisco) has been found in Vicia faba L. guard cell chloroplasts by two immunological methods, using antibodies raised against highly purified subunits of ribulose bisphosphate carboxylase. Indirect cytoimmunofluorescence revealed binding of antibodies against both the small and the large subunits of ribulose bisphosphate carboxylase. Binding was observed only after partial digestion of guard cell walls by 4% Cellulysin to facilitate antibody penetration. After electrophoresis of a homogenate of guard cell protoplasts, the presence of both subunits was also revealed by immunolabeling technique. Positive response required the inhibition of proteolysis which appeared to be active upon homogenization.     

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.     

Protective roles for ATM in cellular response to oxidative stress

ATM (ataxia telangiectasia mutated), the gene mutated in ataxia telangiectasia, is related to a family of large phosphatidylinositol 3-kinase domain-containing proteins involved in cell cycle control and DNA repair. We found that ATM(-/-) DT40 cells were more susceptible than wild-type cells to apoptosis induced not only by ionizing radiation and bleomycin but also by non-DNA-damaging apoptotic stimuli such as C(2)-ceramide. Furthermore, the apoptosis induced by C(2)-ceramide and H(2)O(2) was blocked by anti-oxidants, indicating that the ATM(-/-) DT40 cells had a heightened susceptibility to apoptosis induced by reactive oxygen intermediates (ROI), presumably due to defective ROI-detoxification activities. In support of this hypothesis, we found that more ROI were generated in ATM(-/-) DT40 cells than in wild-type cells, following treatment with the above apoptotic stimuli. These results indicate that ATM plays important roles in the maintenance of the cell homeostasis in response to oxidative damage.     

Salicylic acid negatively affects the response to salt stress in pea plants

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 μm) and glutathione-S transferase (GST; 50 μm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 μm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.     

Immunological evidence for the presence of myelin-related integral proteins in the CNS of hagfish and lamprey

Antibodies against myelin proteins were utilized in the analysis of total particulate material from the brains of the agnathan hagfish and lamprey. Immunoblotting revealed in both species the presence of bands at 50,000 dalton that reacted with anti-bovine PNS-P₀ antibodies. Single bands of 34,000 dalton and 51,000 dalton were immunodetected with antitrout CNS-36K antibodies in lamprey and hagfish, respectively. Antibodies against mammalian myelin basic protein (MBP) and proteolipid protein (PLP) were not recognized. In spite of the lack of multilayered myelin in agnatha, the presence of myelin-related integral proteins suggests that agnathan glial cells have already acquired the capacity to synthesize some proteins that are similar to typical myelin proteins. This represents a crucial evolutionary step towards myelination.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Telomerase activity in response to mild oxidative stress

We have analysed telomerase activity to determine whether it can be modified when BCL-2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl-2 overexpression due to a retroviral infection. Endogenous bcl-2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl-2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl-2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL-2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL-2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL-2 expression when treating patients with ROS (reactive oxygen species) generation by anti-cancer drugs.