Tyrosine Nitration Research Articles

Nitric Oxide Is Essential to Keep the Postharvest Quality of Fruits and Vegetables

Nitric oxide (NO) is a gaseous free radical that has been become a potential tool to maintain the quality of postharvest horticultural produce. It plays important roles in delaying ripening, alleviating chilling injury, preventing browning, and enhancing disease resistance. The regulatory function of NO is achieved through the post-transcriptional modification of proteins, such as tyrosine nitration, S-nitrosylation, and nitroalkylation. Secondly, NO can also induce the expression of stress-related genes by synergistically interacting with other signaling substances, such as Ca2+, ethylene (ETH), salicylic acid (SA), and jasmonic acid (JA). Here, research progress on the role of NO and its donors in regulating the quality of postharvest fruits and vegetables under storage is reviewed. The function of NO crosstalk with other phytohormones is summarized. Future research directions for NO commercial application and the endogenous NO regulatory mechanism are also discussed.

Free Radicals Mediated Redox Signaling in Plant Stress Tolerance.

Abiotic and biotic stresses negatively affect plant cellular and biological processes, limiting their growth and productivity. Plants respond to these environmental cues and biotrophic attackers by activating intricate metabolic-molecular signaling networks precisely and coordinately. One of the initial signaling networks activated is involved in the generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS). Recent research has exemplified that ROS below the threshold level can stimulate plant survival by modulating redox homeostasis and regulating various genes of the stress defense pathway. In contrast, RNS regulates the stress tolerance potential of crop plants by modulating post-translation modification processes, such as S-nitrosation and tyrosine nitration, improving the stability of protein and DNA and activating the expression of downstream stress-responsive genes. RSS has recently emerged as a new warrior in combating plant stress-induced oxidative damage by modulating various physiological and stress-related processes. Several recent findings have corroborated the existence of intertwined signaling of ROS/RNS/RSS, playing a substantial role in crop stress management. However, the molecular mechanisms underlying their remarkable effect are still unknown. This review comprehensively describes recent ROS/RNS/RSS biology advancements and how they can modulate cell signaling and gene regulation for abiotic stress management in crop plants. Further, the review summarizes the latest information on how these ROS/RNS/RSS signaling interacts with other plant growth regulators and modulates essential plant functions, particularly photosynthesis, cell growth, and apoptosis.

Phytotoxicity evaluation of nutrient-fortified pomegranate peel powders prepared from food waste and their feasibility as biofertilizers

AbstractPomegranate peel powder (PPP) is increasingly used as a bioadsorbent to decontaminate wastewaters due to its adsorptive characteristics. The application of nutrient-fortified bioadsorbents as alternatives to chemical fertilizers can provide an innovative and eco-friendly approach for sustainable waste management. Nevertheless, there is extremely limited information regarding their effects on the growth of agricultural crops. We investigated the effects of raw and nutrient-fortified PPPs on oilseed rape (Brassica napus). Our results showed that the concentration-dependent in vitro phytotoxicity of high PPP doses (germination indices were 109.6%, 63.9%, and 8.9% at the applied concentrations of 0.05%, 0.5%, and 5%) was diminished by the application of nutrient-fortified PPPs (germination indices were 66.0–83.4% even at the highest doses). In pot experiments, most PPP treatments (especially Raw-PPP and the mixture of N- and P-fortified PPPs) promoted the development of aboveground plant parts. Reorganization of the pattern of protein tyrosine nitration in the root tissues indicated that the plants were acclimated to the presence of PPPs, and thus, PPP treatment induced no or low-level stress. Our findings confirmed that several doses of PPP supplementation were beneficial for the model crop plant when applied in soil. We anticipate that our study will be a foundation for future investigations involving more plant species and soil types, which can contribute to the introduction of nutrient-fortified PPPs as sustainable biofertilizers.

Complete and selective nitration of tyrosine residue in peptides caused by ultraviolet matrix-assisted laser desorption/ionization.

Complete and highly selective nitration of tyrosine (Tyr) as a residue-specific modification in peptides was found without side reactions, using ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) with a nitroaromatic reagent 3, 5-dinitrosalicylic acid (3,5-DNSA). The tyrosine nitration supported two propositions, namely, the UV-induced. NO2 attack reaction mechanism by Long et al. and the C-NO2 homolysis as a thermal process by Wiik et al. and Furman et al. With the UV-MALDI of peptides, a residue-specific reaction was observed in glycine (Gly) residue, i.e., an oxidation of the alpha-carbon of Gly due to attack of hydroxyl radical (.OH).

Abstract 15194: Nitrative Modification of Caveolin-3, a Novel Mechanism Revealing Cardiac Insulin Resistance in Ischemic Heart Failure

Introduction: Myocardial insulin resistant is a hallmark of diabetic cardiac injury. However, underlying molecular mechanisms remain unclear. Recent studies show that diabetic heart is resistant to other cardioprotective interventions, including adiponectin and pre-conditioning. The “universal” resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad pro-survival signaling cascades. Caveolin (Cav) is a scaffolding protein coordinating transmembrane signaling transduction. However, role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure (HF) is unknown. Methods and Results: Mice were fed normal diet (ND) or high-fat-diet (HFD) and subjected to myocardial ischemia and reperfusion. The cardioprotective effect of insulin was significantly blunted as early as 4 weeks of HFD feeding (pre-diabetes), when insulin signals remain unchanged. However, Cav3/IRβ complex formation, requisite for insulin transmembrane signaling, was significantly reduced. Among multiple post-translational modifications altering proteins interaction, Cav3 (not IRβ or AdipoR1) tyrosine nitration is prominent in the pre-diabetic heart. SIN-1 treatment reduced the signalsome formation and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr 73 is the Cav3 nitration site. Phenylalanine substitution of Tyr 73 (Cav3 Y73F ) abolished SIN-1 induced Cav3 nitration, restored Cav3/IRβ complex, and rescued insulin transmembrane signaling. AAV9-mediated cardiac Cav3 Y73F re-expression blocked HFD-induced Cav3 nitration, preserved Cav3 signalsome integrity, and rescued insulin protective action against ischemic HF. Finally, diabetic nitrative modification of Cav3 at Tyr 73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. Conclusion: Nitration of Cav3 at Tyr 73 and resultant signal complex dissociation is responsible for cardiac insulin/adiponectin resistance in the pre-diabetic heart, contributing to ischemic HF progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic HF.

Hidden networks of aberrant protein transnitrosylation contribute to synapse loss in Alzheimer's disease.

Emerging evidence indicates the importance of S-nitrosation in regulating protein function and activity. This chemical reaction has been termed protein S-nitrosylation to emphasize its biological importance as a posttranslational modification, in some ways reminiscent of phosphorylation. The reaction at cysteine thiols is distinct from other chemical reactions of nitric oxide (NO) that activate soluble guanylate cyclase via nitrosylation of heme or formation of peroxynitrite via reaction with superoxide anion to produce tyrosine nitration. Here, we review the importance of pathological, aberrant transnitrosylation reactions, i.e., transfer of the NO group from one protein to another, and its consequent effect on the pathogenesis of neurological disorders, to date on Alzheimer's disease (AD), but also expected to affect Parkinson's disease (PD)/Lewy body dementia (LBD), HIV-associated neurocognitive disorder (HAND), and other neurodegenerative and neurodevelopmental disorders.

Site-Specific Tyrosine Nitration of Heat Shock Protein 90 Plays Distinct Roles in Glioblastoma Multiforme

Site-Specific Tyrosine Nitration of Heat Shock Protein 90 Plays Distinct Roles in Glioblastoma Multiforme

Effects of high glutamate concentrations on mitochondria of human neuroblastoma SH-SY5Y cells

The excess amount of glutamate in neurons is associated with the excitotoxicity and neurodegenerative diseases. Glutamate induces neurotoxicity primarily by immense influx of Ca2+arising from overstimulation of the NMDA subtype of glutamate receptors. The neuronal death induced by the overstimulation of glutamate receptors depends critically on a sustained increase in mitochondrial Ca2+influx and impairment in mitochondrial functions. The mitochondrial impairment is an important contributor to the glutamate-induced neuronal toxicity and thus provides an important target for the intervention. The present study investigates the effects of high glutamate concentrations on mitochondrial functions. Here, we have shown that the higher concentration of glutamate treatment caused a significant elevation in the N-methyl-D-aspartate (NMDA) receptors expression and elevated the intra-mitochondrial calcium accumulation in SHSY5Y neuronal cells. As a result of an accumulation of intra-mitochondrial calcium, there is a concentration-dependent elevation in ROS in the mitochondria. Tyrosine nitration of several mitochondrial proteins was increased while the mitochondrial membrane potential was dissipated. Furthermore, glutamate treatments also resulted in mitochondrial membrane permeability transition. These findings suggest that treatment of high glutamate concentration causes impairment of mitochondrial functions by an increase in intra-mitochondrial calcium, ROS production, dissipation of mitochondrial membrane potential and mitochondrial permeability transition pore opening in human neuroblastoma SHSY5Y cells.

Catalase post-translational modifications as key targets in the control of erythrocyte redox homeostasis in children with obesity and insulin resistance.

Insulin resistance (IR) is the most common metabolic disturbance in children with obesity. Children with obesity and insulin resistance (ObIR+) display a detriment in erythroid antioxidant defenses, caused by an impaired catalase activity and the increase in oxidative and pro-inflammatory markers. Therefore, erythrocytes from ObRI+are more vulnerable to any oxidative stress elicitor. Since catalase is one of the erythrocytes' first antioxidant defenses, we intended to delve into the mechanisms underlying catalase's impaired activity. Given the lack of cellular organelles in erythrocytes, which prevents protein synthesis, we aimed study catalase post-translational modifications (PTMs) as targets of pro-inflammatory and pro-oxidant status of these cells in children with obesity and IR. Catalase levels of O-glycosylation, tyrosine nitration and S-glutathionylation were analyzed by Western blotting (WB) using immunoprecipitated catalase (IP-CAT) from erythrocyte lysates. Furthermore, Catalase was also identified by LC-MS/MS after isolation and enrichment of erythrocyte nitrosated proteins with a biotin switch approach. The results obtained suggest that catalase inhibition seen in children with obesity is partly due to the increase in the S-nitrosation of the enzyme. Indeed, exogenous administration of nitric oxide (NO) to cultured erythrocytes resulted in a decrease in catalase activity in all groups. Signals of other PTMs (O-glycosylation, Tyr-nitration and S-glutathionylation) were also detected in the erythrocyte catalase in every groups, although levels of catalase O-glycosylation and S-glutathionylation decreased in ObIR+. No evidence of differences in Tyr-nitration of catalase levels were found among groups. The study again highlights the role of erythrocytes as sensors of the inflammatory and pro-oxidant response to which these cells are subjected in children with obesity and insulin resistance.

Limited Zn supply affects nutrient distribution, carbon metabolism and causes nitro-oxidative stress in sensitive Brassica napus

Despite being a worldwide problem, responses of crops to zinc (Zn) deficiency is largely unknown. This study examines the effects of limited Zn supply in Brassica napus . The decreased Zn content of the nutrient solution caused reduced tissue Zn concentrations and reduced K, Ca, Mg levels were detected in the leaves. For Ca, Zn, Fe, Mo inhomogeneous distribution was induced by Zn limitation. Suboptimal Zn supply decreased the shoot and root biomass and altered the root structure. The levels of sugars and sugar phosphates (e.g. glucose, glucose-6-phosphate etc.) were decreased suggesting disturbance in carbon metabolism. Limited Zn availability induced organ-dependent superoxide anion and hydrogen peroxide production and triggered altered NADPH oxidase, superoxide dismutase activities, ascorbate peroxidase abundance and ascorbate and glutathione contents indicating oxidative stress. Moreover, nitric oxide, peroxynitrite and S-nitrosoglutathione (GSNO) levels and GSNO reductase gene expression, protein level and activity were modified by Zn limitation. As a consequence, protein tyrosine nitration and protein carbonylation were intensified in B. napus grown with suboptimal Zn supply. Collectively, these results provide the first evidence for Zn deficiency-induced imbalances in nutrient status, sugar contents, reactive oxygen and nitrogen species metabolisms and for the secondary nitro-oxidative stress in sensitive Brassica napus . • Brassica napus is sensitive to Zn limitation. • Zn deficiency is accompanied by disturbed nutrient homeostasis in Brassica leaves. • Zn limitation disturbs carbon metabolism indicated by reduced sugar contents. • Zn deficiency induces oxidative stress due to disturbed antioxidant defence. • Zn limitation disturbs reactive nitrogen species levels leading to nitro-oxidative stress.

Uncommon posttranslational modifications in proteomics: ADP-ribosylation, tyrosine nitration, and tyrosine sulfation.

Posttranslational modifications (PTMs) are covalent modifications of proteins that modulate the structure and functions of proteins and regulate biological processes. The development of various mass spectrometry-based proteomics workflows has facilitated the identification of hundreds of PTMs and aided the understanding of biological significance in a high throughput manner. Improvements in sample preparation and PTM enrichment techniques, instrumentation for liquid chromatography-tandem mass spectrometry (LC-MS/MS), and advanced data analysis tools enhance the specificity and sensitivity of PTM identification. Highly prevalent PTMs like phosphorylation, glycosylation, acetylation, ubiquitinylation, and methylation are extensively studied. However, the functions and impact of less abundant PTMs are not as well understood and underscore the need for analytical methods that aim to characterize these PTMs. This review focuses on the advancement and analytical challenges associated with the characterization of three less common but biologically relevant PTMs, specifically, adenosine diphosphate-ribosylation, tyrosine sulfation, and tyrosine nitration. The advantages and disadvantages of various enrichment, separation, and MS/MS techniques utilized to identify and localize these PTMs are described.

A perspective view of nitric oxide: biosynthesis, metabolism and signaling in plants

<p indent="0mm">Nitric oxide (NO) is an important gaseous signaling molecule widely distributed in living organisms, it modulates protein stability and enzyme activities mainly through post-translational modifications, including <italic>S</italic>-nitrosylation and tyrosine nitration. NO regulates multiple physiological processes during plant development and stress responses. Moreover, the complex interplay among NO and phytohormones, reactive oxygen species and other signaling molecules precisely regulates various stages of plant growth and development to maintain normal life activities. Here, we review the synthesis and metabolism of NO, and provide insights into the mechanism and biological significance of NO in the regulation of plant development and stress responses.

Nitration of Tyrosine by the Action of Pulsed Radiation of Spark-Discharge Hot Plasma

Nitration of Tyrosine by the Action of Pulsed Radiation of Spark-Discharge Hot Plasma

Variation in glucose metabolism under acidified sodium nitrite mediated nitrosative stress in Saccharomyces cerevisiae.

The work aimed to understand the important changes during glucose metabolism in Saccharomyces cerevisiae under acidified sodium nitrite (ac.NaNO2 ) mediated nitrosative stress. Confocal microscopy and fluorescence-activated cell sorting analysis were performed to investigate the generation of reactive nitrogen and oxygen species, and redox homeostasis under nitrosative stress was also characterized. Quantitative PCR analysis revealed that the expression of ADH genes was upregulated under such condition, whereas the ACO2 gene was downregulated. Some of the enzymes of the tricarboxylic acid cycle were partially inhibited, whereas malate metabolism and alcoholic fermentation were increased under nitrosative stress. Kinetics of ethanol production was also characterized. A network analysis was conducted to validate our findings. In the presence of ac.NaNO2 , in vitro protein tyrosine nitration formation was checked by western blotting using pure alcohol dehydrogenase and aconitase. Alcoholic fermentation rate was increased under stress condition and this altered metabolism might be conjoined with the defence machinery to overcome the nitrosative stress. This is the first work of this kind where the role of metabolism under nitrosative stress has been characterized in S. cerevisiae and it will provide a base to develop an alternative method of industrial ethanol production.

Tailoring Silica-Based Nanoscintillators for Peroxynitrite-Potentiated Nitrosative Stress in Postoperative Radiotherapy of Colon Cancer.

The development of a manageable reactive nitrogen species-potentiated nitrosative stress induction system for cancer therapy has remained elusive. Herein, tailored silica-based nanoscintillators were reported for low-dosage X-ray boosting for the in situ formation of highly cytotoxic peroxynitrite (ONOO-). Significantly, cellular nitrosative stress revolving around the intracellular protein tyrosine nitration through ONOO- pathways was explored. High-energy X-rays were directly deposited on silica-based nanoscintillators, forming the concept of an open source and a reduced expenditure-aggravated DNA damage strategy. Moreover, the resultant ONOO-, along with the released nitric oxide, not only can act as "oxygen suppliers" to combat tumor hypoxia but also can induce mitochondrial damage to initiate caspase-mediated apoptosis, further improving the therapeutic efficacy of radiotherapy. Thus, the design of advanced nanoscintillators with specific enhanced nitrosative stress offers promising potential for postoperative radiotherapy of colon cancer.

In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.

Raman Fingerprints of the SARS-CoV-2 Delta Variant and Mechanisms of Its Instantaneous Inactivation by Silicon Nitride Bioceramics.

Raman spectroscopy uncovered molecular scale markers of the viral structure of the SARS-CoV-2 Delta variant and related viral inactivation mechanisms at the biological interface with silicon nitride (Si3N4) bioceramics. A comparison of Raman spectra collected on the TY11-927 variant (lineage B.1.617.2; simply referred to as the Delta variant henceforth) with those of the JPN/TY/WK-521 variant (lineage B.1.617.1; referred to as the Kappa variant or simply as the Japanese isolate henceforth) revealed the occurrence of key mutations of the spike receptor together with profound structural differences in the molecular structure/symmetry of sulfur-containing amino acid and altered hydrophobic interactions of the tyrosine residue. Additionally, different vibrational fractions of RNA purines and pyrimidines and dissimilar protein secondary structures were also recorded. Despite mutations, hydrolytic reactions at the surface of silicon nitride (Si3N4) bioceramics induced instantaneous inactivation of the Delta variant at the same rate as that of the Kappa variant. Contact between virions and micrometric Si3N4 particles yielded post-translational deimination of arginine spike residues, methionine sulfoxidation, tyrosine nitration, and oxidation of RNA purines to form formamidopyrimidines. Si3N4 bioceramics proved to be a safe and effective inorganic compound for instantaneous environmental sanitation.

Sirtuin1 inhibitor attenuates hypertension in spontaneously hypertensive rats: role of Giα proteins and nitroxidative stress.

We recently showed that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) exhibit overexpression of Sirtuin1 (Sirt1) that contributes to the enhanced expression of Giα proteins implicated in the development of hypertension in SHR. The present study investigated if the inhibition of Sirt1 could also ameliorate hypertension in SHR and explore the underlying molecular mechanisms. For this study, a selective inhibitor of Sirt1, EX-527 (5 mg/kg of body weight), was injected intraperitoneally into 8-week-old SHR and age-matched Wistar Kyoto (WKY) rats twice per week for 3 weeks. The blood pressure (BP) and heart rate was measured twice a week by the CODA noninvasive tail cuff method. The high BP and augmented heart rate in SHR was significantly attenuated by EX-527 treatment, which was associated with the suppression of the overexpression of Sirt1 and Giα proteins in heart, VSMC and aorta. In addition, the enhanced levels of superoxide anion, NADPH oxidase activity, overexpression of NADPH oxidase subunits and FOXO1 were attenuated and the decreased levels of endothelial nitric oxide synthase (eNOS), nitric oxide and increased levels of peroxynitrite (ONOO-) and tyrosine nitration in VSMC from SHR were restored to control levels by EX-527 treatment. Furthermore, knockdown of FOXO1 by siRNA also attenuated the overexpression of Giα-2 and NADPH oxidase subunit proteins and restored the decreased expression of eNOS in VSMC from SHR. These results suggest that the inhibition of overexpressed Sirt1 and its target FOXO1 through decreasing the enhanced levels of Giα proteins and nitro-oxidative stress attenuates the high BP in SHR.

Influence of plasma halide, pseudohalide and nitrite ions on myeloperoxidase-mediated protein and extracellular matrix damage

Myeloperoxidase (MPO) mediates pathogen destruction by generating the bactericidal oxidant hypochlorous acid (HOCl). Formation of this oxidant is however associated with host tissue damage and disease. MPO also utilizes H2O2 to oxidize other substrates, and we hypothesized that mixtures of other plasma anions, including bromide (Br−), iodide (I−), thiocyanate (SCN−) and nitrite (NO2−), at normal or supplemented concentrations, might modulate MPO-mediated HOCl damage. For the (pseudo)halide anions, only SCN− significantly modulated HOCl formation (IC50 ∼33 μM), which is within the normal physiological range, as judged by damage to human plasma fibronectin or extracellular matrix preparations detected by ELISA and LC-MS. NO2− modulated HOCl-mediated damage, in a dose-dependent manner, at physiologically-attainable anion concentrations. However, this was accompanied by increased tyrosine and tryptophan nitration (detected by ELISA and LC-MS), and the overall extent of damage remained approximately constant. Increasing NO2− concentrations (0.5–20 μM) diminished HOCl-mediated modification of tyrosine and methionine, whereas tryptophan loss was enhanced. At higher NO2− concentrations, enhanced tyrosine and methionine loss was detected. These analytical data were confirmed in studies of cell adhesion and metabolic activity. Together, these data indicate that endogenous plasma levels of SCN− (but not Br− or I−) can modulate protein modification induced by MPO, including the extent of chlorination. In contrast, NO2− alters the type of modification, but does not markedly decrease its extent, with chlorination replaced by nitration. These data also indicate that MPO could be a major source of nitration in vivo, and particularly at inflammatory sites where NO2− levels are often elevated.

2-Methoxyethyl Nitrite as a Reagent for Chemoselective On-Water Nitration.

An on-water approach has been developed that allows a nitration of tyrosines and phenols under mild conditions. We envisioned that the assembly of tyrosine/tyrosyl radical couples with interfacial water molecules would realize a biomimetic stacking hydrogen atom transfer (HAT) transition state to facilitate the electron-transfer process. The optimal organic nitrite, 2-methoxyethyl nitrite, resulted in rapid coupling of the tyrosyl radicals with ⋅NO2 at the oil-water interface to afford the nitrated phenols. Many characteristics found in our on-water strategy are distinct from other complementary systems that include radical nitration. These enticing roles of water in the reaction process introduce new avenues to explore in the design of synthetic organic chemistry systems.