Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis.

Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low- to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions, a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer.

Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These posttranslational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation, and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.

Mass spectrometry analysis of phosphotyrosine-containing proteins.

Tyrosine phosphorylation is a crucial posttranslational modification that is involved in various aspects of cell biology and often has functions in cancers. It is necessary not only to identify the specific phosphorylation sites but also to quantify their phosphorylation levels under specific pathophysiological conditions. Because of its high sensitivity and accuracy, mass spectrometry (MS) has been widely used to identify endogenous and synthetic phosphotyrosine proteins/peptides across a range of biological systems. However, phosphotyrosine-containing proteins occur in extremely low abundance and they degrade easily, severely challenging the application of MS. This review highlights the advances in both quantitative analysis procedures and enrichment approaches to tyrosine phosphorylation before MS analysis and reviews the differences among phosphorylation, sulfation, and nitration of tyrosine residues in proteins. In-depth insights into tyrosine phosphorylation in a wide variety of biological systems will offer a deep understanding of how signal transduction regulates cellular physiology and the development of tyrosine phosphorylation-related drugs as cancer therapeutics.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

Far-UVC 222 nm Treatment: Effects of Nitrate/Nitrite on Disinfection Byproduct Formation Potential.

Irradiation at far ultraviolet C (far-UVC) 222 nm by krypton chloride (KrCl*) excilamps can enhance microbial disinfection and micropollutant photolysis/oxidation. However, nitrate/nitrite, which absorbs strongly at 222 nm, may affect the formation of disinfection byproducts (DBPs). Herein, we evaluated model organic matter and real water samples and observed a substantial increase in the formation potential for trichloronitromethane (chloropicrin) (TCNM-FP), a nitrogenous DBP, by nitrate or nitrite after irradiation at 222 nm. At a disinfection dose of 100 mJ·cm-2, TCNM-FP of humic acids and fulvic acids increased from ∼0.4 to 25 and 43 µg·L-1, respectively, by the presence of 10 mg-N·L-1 nitrate. For the effect of nitrate concentration, the TCNM-FP peak was observed at 5-10 mg-N·L-1. Stronger fluence caused a greater increase of TCNM-FP. Similarly, the increase of TCNM-FP was also observed for wastewater and drinking water samples containing nitrate. Pretreatment using ozonation and coagulation, flocculation, and filtration or the addition of H2O2 can effectively control TCNM-FP. The formation potential of other DBPs was minorly affected by irradiation at 222 nm regardless of whether nitrate/nitrite was present. Overall, far-UVC 222 nm treatment poses the risk of increasing TCNM-FP of waters containing nitrate or nitrite at environmentally relevant concentrations and the mitigation strategies merit further research.

Tyrosine nitration enhances the allergenic potential of house dust mite allergen Der p 2.

Nitration of allergenic proteins caused by atmospheric pollutants O3 and NO2 may enhance their allergenic potential. In the study, the influence of nitration was investigated on the allergenicity of Der p 2, which is a main allergen from house dust mites and plays an important role in allergenic rhinitis and asthma. The results reveal that nitrated Der p 2 enhanced the IgE-binding capacity, upregulated the mRNA expression and release of IL-6 and IL-8 from bronchial epithelial cells, and induced higher levels of specific-IgE, TH2 cytokines and white blood cells in mice. Besides, nitrated Der p 2 caused more severe oxidative stress and allergenic symptoms in mice. It is concluded that nitration enhanced the allergenicity of Der p 2 through not only directly inducing higher amount of specific-IgE and stronger responses of TH2 cytokines, but also indirectly aggravating allergic symptoms by oxidative stress and adjuvant-like activation airway epithelial cells. The study suggests that the contribution of nitration to the promotion in allergenicity should not be ignored when precisely assessing the risk of house dust mite allergens in real environment.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Convenient Preparation, Thermal Properties and X-ray Structure Determination of 2,3-Dihydro-5,6,7,8-tetranitro-1,4-benzodioxine (TNBD): A Promising High-Energy-Density Material.

2,3-dihydro-5,6,7,8-tetranitro-1,4-benzodioxine (TNBD), molecular formula = C8H4N4O10, is a completely nitrated aromatic ring 1,4-benzodioxane derivative. The convenient method of TNBD synthesis was developed (yield = 81%). The detailed structure of this compound was investigated by X-ray crystallography. The results of the thermal analysis (TG) obtained with twice re-crystallized material revealed the onset at 240 °C (partial sublimation started) and melting at 286 °C. The investigated material degraded completely at 290-329 °C. The experimental density of 1.85 g/cm3 of TNBD was determined by X-ray crystallography. The spectral properties of TNBD (NMR, FT-IR and Raman) were explored. The detonation properties of TNBD calculated by the EXPLO 5 code were slightly superior in comparison to standard high-energy material-tetryl (detonation velocity of TNBD-7727 m/s; detonation pressure-278 kbar; and tetryl-7570 m/s and 226.4 kbar at 1.614 g/cm3, or 260 kbar at higher density at 1.71 g/cm3. The obtained preliminary results might suggest TNBD can be a potential thermostable high-energy and -density material (HEDM).