Departamento de Biología Experimental

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Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases
(OXFORD UNIV PRESS, 2020-01) Matamoros, Manuel Ángel; Cutrona, María C.; Wienkoop, Stefanie; Begara-Morales, Juan Carlos; Sandal, Niels; Orera, Irene; Barroso-Albarracín, Juan Bautista; Stougaard, Jens; Becana, Manuel
Nitric 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-nitrosyla tion. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cas cades 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 glutathionyla tion 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 develop mental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in leg ume biology.
Differential modulation of S-nitrosoglutathione reductase and reactive nitrogen species in wild and cultivated tomato genotypes during development and powdery mildew infection.
(ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2020-10) Jahnová, Jana; Činčalová, Lucie; Sedlářová, Michaela; Jedelská, Tereza; Sekaninová, Jana; Mieslerová, Barbora; Luhová, Lenka; Barroso-Albarracín, Juan Bautista ; Petřivalský, Marek
Nitric oxide plays an important role in the pathogenesis of Pseudoidium neolycopersici, the causative agent of tomato powdery mildew. S-nitrosoglutathione reductase, the key enzyme of S-nitrosothiol homeostasis, was investigated during plant development and following infection in three genotypes of Solanum spp. differing in their resistance to P. neolycopersici. Levels and localization of reactive nitrogen species (RNS) including NO, S nitrosoglutathione (GSNO) and peroxynitrite were studied together with protein nitration and the activity of nitrate reductase (NR). GSNOR expression profiles and enzyme activities were modulated during plant devel opment and important differences among Solanum spp. genotypes were observed, accompanied by modulation of NO, GSNO, peroxynitrite and nitrated proteins levels. GSNOR was down-regulated in infected plants, with exception of resistant S. habrochaites early after inoculation. Modulations of GSNOR activities in response to pathogen infection were found also on the systemic level in leaves above and below the inoculation site. Infection strongly increased NR activity and gene expression in resistant S. habrochaites in contrast to susceptible S. lycopersicum. Obtained data confirm the key role of GSNOR and modulations of RNS during plant development under normal conditions and point to their involvement in molecular mechanisms of tomato responses to bio trophic pathogens on local and systemic levels.
Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation
(Oxford Academic, 2015-06-25) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla-Serrano, María Nieves; López-Jaramillo, Javier; Luque-Vázquez, Francisco; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
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 NADP-binding 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 (150mM 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.
Differential transcriptomic analysis by RNA-Seq of GSNO-responsive genes between Arabidopsis roots and leaves
(Oxford Academic, 2014-06) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leyva-Pérez, María O; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
S-Nitrosoglutathione (GSNO) is a nitric oxide-derived molecule that can regulate protein function by a post-translational modification designated S-nitrosylation. GSNO has also been detected in different plant organs under physiological and stress conditions, and it can also modulate gene expression. Thirty-day-old Arabidopsis plants were grown under hydroponic conditions, and exogenous 1 mM GSNO was applied to the root systems for 3 h. Differential gene expression analyses were carried out both in roots and in leaves by RNA sequencing (RNA-seq). A total of 3,263 genes were identified as being modulated by GSNO. Most of the genes identified were associated with the mechanism of protection against stress situations, many of these having previously been identified as target genes of GSNO by array-based methods. However, new genes were identified, such as that for methionine sulfoxide reductase (MSR) in leaves or different miscellaneous RNA (miscRNA) genes in Arabidopsis roots. As a result, 1,945 GSNO-responsive genes expressed differently in leaves and roots were identified, and 114 of these corresponded exclusively to one of these organs. In summary, it is demonstrated that RNA-seq extends our knowledge of GSNO as a signaling molecule which differentially modulates gene expression in roots and leaves under non-stress conditions.
Drought stress triggers the accumulation of NO and SNOs in cortical cells of Lotus japonicus L. roots and the nitration of proteins with relevant metabolic function
(Elsevier, 2018-08-16) Signorelli, Santiago; Corpas, Francisco Javier; Rodríguez-Ruiz, Marta; Valderrama, Raquel; Barroso-Albarracín, Juan Bautista; Borsani, Omar; Monza, Jorge
Drought is considered one of the abiotic stresses with significant implications on plant productivity. Previously, we have shown that water deficit produces a differential nitro-oxidative stress in roots and leaves of Lotus japonicus L. plants. Using this model legume, we studied the nitro-oxidative stress in drought-stressed roots by complementary biochemical, cellular and proteomic approaches. Cellular analyses of root cross-sections by confocal laser scanning microscopy (CLSM) using specific fluorescent probes for superoxide radical (O2·−), nitric oxide (NO), peroxynitrite (ONOO−) and S-nitrosothiols (SNOs) showed that drought stress causes a differential cellular localization of these reactive species. Mainly, O2·− and ONOO− had a wide distribution in almost all root cell types (xylem, parenchyma, and peridermis), whereas NO and SNOs accumulated in cortical cells (peridermis). Liquid chromatography-electrospray/mass spectrometry (LC-ES/MS) analyses showed that the content of ascorbate, S-nitrosoglutaathione (GSNO), and reduced glutathione (GSH) in drought-stressed roots was drastically diminished. Nitroproteome analysis by two-dimensional gel electrophoresis and mass spectrometry allowed to identify 13 tyrosine-nitrated proteins such as methionine synthase, Hsp70, adenosyl-homocysteinase, peroxidase, alcohol dehydrogenases, glutamine synthetase, fructokinase, 1,3-beta-glucanase, chitinases, endochitinase, among others which are directly (24%) or indirectly (74%) related to plant defense. Taken together, these results indicate that drought-stressed roots have an active metabolism of reactive oxygen and nitrogen species (ROS and RNS) characterized by an increase of protein nitration and accumulation of NO and SNOs in cortical cells. The possibility of autophagy taking place in the stressed roots is also discussed.
Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation
(OXFORD UNIV PRESS, 2014-02) Begara-Morales, Juan Carlos; Sanchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; López-Jaramillo, Jaime; Padilla-Serrano, María Nieves; Carreras, Alfonso; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150 mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H₂O₂, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.
Endogenous Biosynthesis of S-Nitrosoglutathione From Nitro-Fatty Acids in Plants
(FRONTIERS MEDIA SA, 2020-06) Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Sánchez-Calvo, Beatriz; Begara-Morales, Juan Carlos; Valderrama, Raquel; Chaki, Mounira; Aranda-Caño, Lorena; Moreno-González, David; Molina-Díaz, Antonio; Barroso-Albarracín, Juan Bautista
Nitro-fatty acids (NO2-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 NO2-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO2-FAs highlight the ability to release NO therefore modulating specific protein targets through post translational modifications (NO-PTMs). Thus, based on the capacity of NO2-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO2-Ln) can modulate S nitrosoglutathione (GSNO) biosynthesis in Arabidopsis. The incubation of NO2-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 15N-labeled NO2-Ln throughout the roots, and 15N-labeled GSNO (GS15NO) was detected in the leaves. With the aim to go in depth in the relation of NO2-FA and GSNO in plants, Arabidopsis alkenal reductase mutants (aer mutants) which modulate NO2-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 NO2-FAs, increasing knowledge about S-nitrosothiols and GSNO signaling pathways in plants.
High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration
(WILEY, 2011-06) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; López-Jaramillo, Jaime; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO2-Tyr) studied by highperformance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin–NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.
Hydrogen sulfide: A novel component in Arabidopsis peroxisomes which triggers catalase inhibition
(WILEY, 2019-07) Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista; González-Gordo, Salvador; Muñoz-Vargas, María Ángeles; Palma, José Manuel
Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H2O2, superoxide radical (O2), nitric oxide and peroxynitrite (ONOO-). These organelles have an active nitro oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H2S) is a new signaling gasotransmitter which can mediate the posttranslational modification (PTM) persulfidation. We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) fused to a canonical peroxisome targeting signal 1 (PTS1) to visualize peroxisomes in living cells, as well as a specific fluorescent probe which showed that peroxisomes contain H2S. H2S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H2O2-generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H2S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H2S donor. To corroborate the inhibitory effect of H2S on catalase activity, we also assayed pure catalase from bovine liver and pepper fruit-enriched samples, in which catalase activity was inhibited. Taken together, these data provide evidence of the presence of H2S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H2O2 metabolism.
Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings
(OXFORD UNIVERSITY PRESS, 2011-03) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-argininedependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a downregulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.
Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions
(OXFORD UNIV PRESS, 2008-09-18) Corpas, Francisco Javier; Chaki, Mounira; Fernández-Ocaña, Ana; Valderrama, Raquel; Palma, José Manuel; Carreras, Alfonso; Begara-Morales, Juan Carlos; Airaki, Morad; del-Río, Luis Alfonso; Barroso-Albarracín, Juan Bautista
Nitric oxide (NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of L-arginine dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO2-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total _NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO2-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.
Metabolism of reactive oxygen species and reactive nitrogen species in pepper (Capsicum annuum L.) plants under low temperature stress
(WILEY, 2012-02) Airaki, Morad; Leterrier, Marina; Mateos, Rosa María; Valderrama, Raquel; Chaki, Mounira; Barroso-Albarracín, Juan Bautista; del-Río, Luis Alfonso; Palma, José Manuel; Corpas, Francisco Javier
Low temperature is an environmental stress that affects crop production and quality and regulates the expression of many genes, and the level of a number of proteins and metabolites. Using leaves from pepper (Capsicum annum L.) plants exposed to low temperature (8°C) for different time periods (1 to 3d), several key components of the metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were analysed. After 24h of exposure at 8°C, pepper plants exhibited visible symptoms characterized by flaccidity of stems and leaves. This was accompanied by significant changes in the metabolism of RNS and ROS with an increase of both protein tyrosine nitration (NO2-Tyr) and lipid peroxidation, indicating that low temperature induces nitrosative and oxidative stress. During the second and third days at low temperature, pepper plants underwent cold acclimation by adjusting their antioxidant metabolism and reverting the observed nitrosative and oxidative stress. In this process, the levels of the soluble non-enzymatic antioxidants ascorbate and glutathione, and the activity of the main NADPH-generating dehydrogenases were significantly induced. This suggests that ascorbate, glutathione and the NADPH-generating dehydrogenases have a role in the process of cold acclimation through their effect on the redox state of the cell.
Nitrated Fatty-Acids Distribution in Storage Biomolecules during Arabidopsis thaliana Development
(MDPI, 2022-09) Valderrama, Raquel; Chaki, Mounira; Begara-Morales, Juan Carlos; Melguizo, Manuel; Barroso-Albarracín, Juan Bautista; Aranda-Caño, Lorena
The non-enzymatic interaction of polyunsaturated fatty acids with nitric oxide (NO) and de rived 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.
Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in Saccharomyces cerevisiae
(MDPI, 2022-05-14) Aranda-Caño, Lorena; Valderrama, Raquel; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Chaki, Mounira; Padilla-Serrano, María Nieves; Melguizo, Manuel; López-Jaramillo, Francisco Javier; Barroso-Albarracín, Juan Bautista
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.
Post-Translational Modification of Proteins Mediated by Nitro-Fatty Acids in Plants: Nitroalkylation
(MDPI, 2019-03-29) Aranda-Caño, Lorena; Sánchez-Calvo, Beatriz; Begara-Morales, Juan Carlos; Chaki, Mounira; Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Valderrama, Raquel; Barroso-Albarracín, Juan Bautista
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–amino acid residues. The adduction of NO2-FAs to protein targets generates a reversible post-translational modification called nitroalkylation. In di erent 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.
Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls
(OXFORD UNIV PRESS, 2009-08-28) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; López-Jaramillo, Jaime; Luque-Vázquez, Francisco; Palma, José Manuel; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María Victoria; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
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 mmol 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.
Protein tyrosine nitration in pea roots during development and senescence
(OXFORD UNIV PRESS, 2013-01-28) Begara-Morales, Juan Carlos; Chaki, Mounira; Sánchez-Calvo, Beatriz; Mata-Pérez, Capilla; Leterrier, Marina; Palma, José Manuel; Barroso-Albarracín, Juan Bautista; Corpas, Francisco Javier
Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence.
Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration
(OXFORD UNIV PRESS, 2015-03-26) Chaki, Mounira; Álvarez-de-Morales, Paz; Ruíz-Torres, Carmelo; Begara-Morales, Juan Carlos; Barroso-Albarracín, Juan Bautista; Corpas, Francisco Javier; Palma, José Manuel
Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.
Role of electrophilic nitrated fatty acids during development and response to abiotic stress processes in plants
(Oxford University Press, 2020-11-07) Begara-Morales, Juan Carlos; Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Chaki, Mounira; Valderrama, Raquel; Aranda-Caño, Lorena; Barroso-Albarracín, Juan Bautista
Nitro-fatty acids are generated from the interaction of unsaturated fatty acids and nitric oxide (NO)-derived molecules. The endogenous occurrence and modulation throughout plant development of nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) suggest a key role for these molecules in initial development stages. In addition, NO2-Ln content increases significantly in stress situations and induces the expression of genes mainly related to abiotic stress, such as genes encoding members of the heat shock response family and antioxidant enzymes. The promoter regions of NO2-Ln-induced genes are also involved mainly in stress responses. These findings confirm that NO2-Ln is involved in plant defense processes against abiotic stress conditions via induction of the chaperone network and antioxidant systems. NO2-Ln signaling capacity lies mainly in its electrophilic nature and allows it to mediate a reversible post-translational modification called nitroalkylation, which is capable of modulating protein function. NO2-Ln is a NO donor that may be involved in NO signaling events and is able to generate S-nitrosoglutathione, the major reservoir of NO in cells and a key player in NO-mediated abiotic stress responses. This review describes the current state of the art regarding the essential role of nitro-fatty acids as signaling mediators in development and abiotic stress processes.
Short-Term Low Temperature Induces Nitro-Oxidative Stress that Deregulates the NADP-Malic Enzyme Function by Tyrosine Nitration in Arabidopsis thaliana
(MDPI, 2019-10-01) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María Victoria; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; López-Jaramillo, Francisco Javier; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
Low temperature (LT) negatively a ects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS).AmongRNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could a ect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the e ect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress a ects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.

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Examinando Departamento de Biología Experimental por Autor "Barroso-Albarracín, Juan Bautista"