Análisis de la nitración fisiológica de proteínas en órganos de plantas de guisante (Pisum sativum L) durante su desarrollo y senescencia

Comunicaciones a congreso

Nitroproteínas implicadas en la maduración de frutos y en el estrés por baja temperatura en plantas de pimiento (Capsicum annuum L)

Comunicaciones a congreso

Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADPbinding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150 mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)- PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.Spanish GovernmentERDF - Ministry of Economy and Competitiveness BIO2012-33904Junta de Andalucía BIO286 BIO19

Oxidative Stress in Plants

Environmental stresses negatively affect plant growth, development and crop productivity [...

Nitrated Fatty-Acids Distribution in Storage Biomolecules during <i>Arabidopsis thaliana</i> Development

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

Post-Translational Modification of Proteins Mediated by Nitro-Fatty Acids in Plants: Nitroalkylation

Nitrate fatty acids (NO2-FAs) are considered reactive lipid species derived from the non-enzymatic oxidation of polyunsaturated fatty acids by nitric oxide (NO) and related species. Nitrate fatty acids are powerful biological electrophiles which can react with biological nucleophiles such as glutathione and certain protein&#8211;amino acid residues. The adduction of NO2-FAs to protein targets generates a reversible post-translational modification called nitroalkylation. In different animal and human systems, NO2-FAs, such as nitro-oleic acid (NO2-OA) and conjugated nitro-linoleic acid (NO2-cLA), have cytoprotective and anti-inflammatory influences in a broad spectrum of pathologies by modulating various intracellular pathways. However, little knowledge on these molecules in the plant kingdom exists. The presence of NO2-OA and NO2-cLA in olives and extra-virgin olive oil and nitro-linolenic acid (NO2-Ln) in Arabidopsis thaliana has recently been detected. Specifically, NO2-Ln acts as a signaling molecule during seed and plant progression and beneath abiotic stress events. It can also release NO and modulate the expression of genes associated with antioxidant responses. Nevertheless, the repercussions of nitroalkylation on plant proteins are still poorly known. In this review, we demonstrate the existence of endogenous nitroalkylation and its effect on the in vitro activity of the antioxidant protein ascorbate peroxidase

Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in <i>Saccharomyces cerevisiae</i>

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 (NO2-OA) in Saccharomyces cerevisiae 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 NO2-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 NO2-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

Altered plant and nodule development and protein S-Nitrosylation in Lotus japonicus mutants deficient in S-Nitrosoglutathione reductases

51 Pags.- 13 Figs.- 1 Tabl.- 1 Dataset. The definitive version is available at: https://academic.oup.com/pcpNitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.Spanish Ministry of Economy and Competitiveness/State Research Agency-European Regional Developmental Fund [grant AGL2017-85775-R], Aragón Government (A09_17R) and Danish National Research Foundation [grant DNRF79].Peer reviewe