Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has ...been reported.
We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.
Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.
These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.
This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.
Display omitted
•Protein tyrosine nitration is a PTM mediated by NO-derived molecules.•Peroxisomal NADH-dependent HPR activity is a target of tyrosine nitration.•Tyr198 of HPR is the primary site of nitration responsible for the inhibition.
Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is ...scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO2-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3′-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO2-Tyr in hypocotyls was 0.56 μmol mg−1 protein and 0.19 pmol mg−1 protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.
Nitro-fatty acids (NO
2
-FAs) are novel molecules resulting from the interaction of unsaturated fatty acids and nitric oxide (NO) or NO-related molecules. In plants, it has recently been described ...that NO
2
-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO
2
-FAs highlight the ability to release NO therefore modulating specific protein targets through post-translational modifications (NO-PTMs). Thus, based on the capacity of NO
2
-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO
2
-Ln) can modulate
S
-nitrosoglutathione (GSNO) biosynthesis in
Arabidopsis
. The incubation of NO
2
-Ln with GSH was analyzed by LC-MS/MS and the
in vitro
synthesis of GSNO was noted. The
in vivo
confirmation of this behavior was carried out by incubating
Arabidopsis
plants with
15
N-labeled NO
2
-Ln throughout the roots, and
15
N-labeled GSNO (GS
15
NO) was detected in the leaves. With the aim to go in depth in the relation of NO
2
-FA and GSNO in plants,
Arabidopsis
alkenal reductase mutants (
aer
mutants) which modulate NO
2
-FAs levels were used. Our results constitute the first evidence of the modulation of a key NO biological reservoir in plants (GSNO) by these novel NO
2
-FAs, increasing knowledge about
S
-nitrosothiols and GSNO-signaling pathways in plants.
Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen ...peroxide (HâOâ) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells.
NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of ...oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 m smallcapital m NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H₂O₂ content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 m smallcapital m NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.
The cellular and subcellular localization of endogenous nitric oxide (NO(.)) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of ...pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO(.) was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO(.) generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO(.) was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO(.), with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO(.) was also found in these organelles, and the level measured of NO(.) was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO(.) from L-Arg (nitric oxide synthase NOS-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO(.) mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO(.) could be involved in the process of senescence of pea leaves.
NADPH regeneration appears to be essential in the mechanism of plant defence against oxidative stress. Plants contain several NADPH-generating dehydrogenases including isocitrate dehydrogenase ...(NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME). In Arabidopsis seedlings grown under salinity conditions (100 mM NaCl) the analysis of physiological parameters, antioxidant enzymes (catalase and superoxide dismutase) and content of superoxide radical (O2 ∙−), nitric oxide (NO), and peroxynitrite (ONOO-) indicates a process of nitro-oxidative stress induced by NaCl. Among the analysed NADPH-generating dehydrogenases under salinity conditions, the NADP-ICDH showed the maximum activity mainly attributable to the root NADP-ICDH. Thus, these data provide new insights on the relevance of the NADP-ICDH which could be considered as a second barrier in the mechanism of response against the nitro-oxidative stress generated by salinity.
The non-enzymatic interaction of polyunsaturated fatty acids with nitric oxide (NO) and derived species results in the formation of nitrated fatty acids (NO2-FAs). These signaling molecules can ...release NO, reversibly esterify with complex lipids, and modulate protein function through the post-translational modification called nitroalkylation. To date, NO2-FAs act as signaling molecules during plant development in plant systems and are involved in defense responses against abiotic stress conditions. In this work, the previously unknown storage biomolecules of NO2-FAs in Arabidopsis thaliana were identified. In addition, the distribution of NO2-FAs in storage biomolecules during plant development was determined, with phytosterol esters (SE) and TAGs being reservoir biomolecules in seeds, which were replaced by phospholipids and proteins in the vegetative, generative, and senescence stages. The detected esterified NO2-FAs were nitro-linolenic acid (NO2-Ln), nitro-oleic acid (NO2-OA), and nitro-linoleic acid (NO2-LA). The last two were detected for the first time in Arabidopsis. The levels of the three NO2-FAs that were esterified in both lipid and protein storage biomolecules showed a decreasing pattern throughout Arabidopsis development. Esterification of NO2-FAs in phospholipids and proteins highlights their involvement in both biomembrane dynamics and signaling processes, respectively, during Arabidopsis plant development.
Heat stress is one of the abiotic stresses that leads to oxidative stress. To protect themselves, yeast cells activate the antioxidant response, in which cytosolic peroxiredoxin Tsa1 plays an ...important role in hydrogen peroxide removal. Concomitantly, the activation of the heat shock response (HSR) is also triggered. Nitro-fatty acids are signaling molecules generated by the interaction of reactive nitrogen species with unsaturated fatty acids. These molecules have been detected in animals and plants. They exert their signaling function mainly through a post-translational modification called nitroalkylation. In addition, these molecules are closely related to the induction of the HSR. In this work, the endogenous presence of nitro-oleic acid (NO
-OA) in
is identified for the first time by LC-MS/MS. Both hydrogen peroxide levels and Tsa1 activity increased after heat stress with no change in protein content. The nitroalkylation of recombinant Tsa1 with NO
-OA was also observed. It is important to point out that cysteine 47 (peroxidatic) and cysteine 171 (resolving) are the main residues responsible for protein activity. Moreover, the in vivo nitroalkylation of Tsa1 peroxidatic cysteine disappeared during heat stress as the hydrogen peroxide generated in this situation caused the rupture of the NO
-OA binding to the protein and, thus, restored Tsa1 activity. Finally, the amino acid targets susceptible to nitroalkylation and the modulatory effect of this PTM on the enzymatic activity of Tsa1 are also shown in vitro and in vivo. This mechanism of response was faster than that involving the induction of genes and the synthesis of new proteins and could be considered as a key element in the fine-tuning regulation of defence mechanisms against oxidative stress in yeast.
The presence and properties of the enzyme xanthine oxidoreductase (XOR) in peroxisomes from pea (
Pisum sativum L.) leaves were studied using biochemical and immunological methods. The activity ...analysis showed that, in leaf peroxisomes, the superoxide-generating XOR form, xanthine oxidase (XOD), was predominant over the xanthine dehydrogenase form (XDH), with a XDH/XOD ratio of 0.5. However, in crude extracts of pea leaves, the XDH form was more abundant, with a XDH/XOD ratio of 1.6. The native molecular mass of the peroxisomal XOR determined by polyacrylamide gel electrophoresis was 290
kDa. Using western blot assays, we identified an immunoreactive band of 59
kDa that was not affected by the reducing reagent DTT or endogenous proteases. The analysis of pea leaves by electron microscopy immunogold labeling with affinity-purified antibodies against rat XOD confirmed that this enzyme was localized in the matrix of peroxisomes, as well as in chloroplasts and cytosol. In pea plants subjected to abiotic stress by cadmium, the activity of the peroxisomal XOR was reduced, whereas its protein level expression increased. The results confirmed that leaf peroxisomes contain XOR, and suggest that this peroxisomal metalloflavoprotein enzyme is involved in the mechanism of response of pea plants to abiotic stress by heavy metals.
PDF
