737 research outputs found

    Structure-function relationship in S-nitrosoglutathione reductase and the development of fluorogenic pseudo-substrates

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    S-nitrosation is the attachment of a nitric oxide moiety to the thiol side chain of cysteine. S-nitrosoglutathione (GSNO) acts as a bioactive reservoir for NO to maintain an equilibrium in the concentration of NO in the body. Due to this, the study of the enzyme S-nitrosoglutathione reductase has of great interest because of its ability to metabolize GSNO. S-nitrosoglutathione reductase’s activity has been linked to a number of human diseases. Chapter 1 of this thesis presents a proposed allosteric binding domain on GSNOR. Positive cooperativity (sigmoidal deviation) was observed from steady state analysis of GSNOR which indicated an affinity for the binding of GSNO at this site. The presence of such a site was further supported by Molecular docking simulations and HDX-MS which showed that the amino acids Gly321, Lys323, Asn185 and Lys188 interact with molecules bound at this site. Chapter two introduces four reagents that can function as probes or pseudo-substrates for the monitoring of enzymatic activity as well as measuring concentrations of free thiols in vitro and live cells. These reagents are N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2), N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2), N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), and N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG). They are easy to prepare and purity and can be used in various applications

    Tomato root growth inhibition by salinity and cadmium is mediated by S-nitrosative modifications of ROS metabolic enzymes controlled by S-nitrosoglutathione reductase

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    S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.Palacky University in Olomouc [IGA_2019_022

    Biochemical and Functional Studies of S-nitrosoglutathione Reductase and Neutral Sphingomyelinase II

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    S-nitrosation is the covalent attachment of nitric oxide (NO) moiety to cysteine thiol side chain. This reversible modification represents an important mechanism of post-translational regulation for a large number of proteins. In the cellular environment, S-nitrosoglutathione (GSNO) can transfer its NO group to reactive cysteine residues within proteins via transnitrosation reactions. Similarly, S-nitrosated protein can transfer its NO moiety to reduced glutathione (GSH). Due to the existence of this equilibrium, the GSNO metabolizing enzyme GSNO reductase (GSNOR) indirectly drives protein de-nitrosation. To date, aberrant GSNOR activity has been implicated in a large spectrum of human diseases. In this dissertation, we report the synthesis and characterization of O-aminobenzoyl-S-nitrosoglutathione (OAbz-GSNO), a novel fluorogenic substrate for GSNOR. OAbz-GSNO reduction mediated by GSNOR results in significant increases in fluorescence; and this increase in fluorescence is attenuated by GSNOR inhibitor treatment. In addition, OAbz-GSNO is cell membrane permeable and can be used to monitor endogenous GSNOR activity in cultured cells. Overall, our work demonstrates that OAbz-GSNO is a useful tool for assessing GSNOR activity, both in vitro and in cells. Site-directed mutagenesis and kinetic studies conducted using recombinant GSNOR suggest acetylation of Lys101 negatively affects enzyme activity; while computational simulations uncovered a putative allosteric GSNO binding site. We have experimental evidence supporting our model that GSNO binding to this allosteric site enhances GSNOR activity. Both lysine acetylation and allosteric substrate binding represent potential mechanisms involved in the post-translation regulation of GSNOR activity. Neutral sphingomyelinase II (NSMase II) is a mediator of cellular stress response. It catalyzes the hydrolysis of plasma membrane sphingomyelin to generate bioactive ceramide and phosphocholine. This project looks into whether chronic cortisol exposure (as a stressor) affects NSMase II expression/activity. Experimental results demonstrate exposure to cortisol leads to increased cell size, but NSMase II expression and activity are unaffected. However, NSMase II over-expressing cells appear to have less cholesterol in the plasma membrane. Since cholesterol is important for the formation of lipid rafts, these finding suggest that in addition to ceramide generation, modulation of plasma membrane cholesterol content may represent an alternative mechanism by which NSMase II exerts its biological effects

    Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

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    BACKGROUND: S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma. METHODS: Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings. RESULTS: N6022 attenuated AHR (ED(50) of 0.015 ± 0.002 mg/kg; Mean ± SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh. CONCLUSIONS: The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease

    Evidence towards the involvement of nitric oxide in drought tolerance of sugarcane

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    © 2017 Elsevier Masson SAS Exogenous supply of nitric oxide (NO) increases drought tolerance in sugarcane plants. However, little is known about the role of NO produced by plants under water deficit. The aim of this study was to test the hypothesis that drought-tolerance in sugarcane is associated with NO production and metabolism, with the more drought-tolerant genotype presenting higher NO accumulation in plant tissues. The sugarcane genotypes IACSP95-5000 (drought-tolerant) and IACSP97-7065 (drought-sensitive) were submitted to water deficit by adding polyethylene glycol (PEG-8000) in nutrient solution to reduce the osmotic potential to−0.4MPa. To evaluate short-time responses to water deficit, leaf and root samples were taken after 24h under water deficit. The drought-tolerant genotype presented higher root extracellular NO content, which was accompanied by higher root nitrate reductase (NR) activity as compared to the drought-sensitive genotype under water deficit. In addition, the drought-tolerant genotype had higher leaf intracellular NO content than the drought-sensitive one. IACSP95-5000 exhibited decreases in root S-nitrosoglutathione reductase (GSNOR) activity under water deficit, suggesting that S-nitrosoglutathione (GSNO) is less degraded and that the drought-tolerant genotype has a higher natural reservoir of NO than the drought-sensitive one. Those differences in intracellular and extracellular NO contents and enzymatic activities were associated with higher leaf hydration in the drought-tolerant genotype as compared to the sensitive one under water deficit

    Cardioprotective Effects of S-Nitrosothiols in Ischemia- Reperfusion: Role for Mitochondria and Calcium Channels

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    The most important clinical consequence of coronary disease is acute myocardial infarction caused by an occlusion that limits the irrigation to the heart. Although the gold standard treatment is to restore blood flow, this reperfusion causes inherent damage by increasing the size of the infarcted area primarily through the opening of the mitochondrial permeability transition pore (MPTP). The cardioprotective effect of nitric oxide (NO) has been described to operate through S-nitrosylation of several important proteins in the cardiomyocytes such as the calcium channels RyR2 and the L-type Ca2+ channel and mitochondrial proteins, including the MPTP. In this sense, an attractive strategy to prevent the ischemia-reperfusion damage is to increase the bioavailability of endogenous S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of NO through denitrosylation, which would limit the cardioprotective effect of NO. Although inhibition of GSNOR has been studied in different organs, its effects on myocardial reperfusion have not yet been fully elucidated. In this chapter, we review the pathophysiology underlying myocardial reperfusion injury and the opening of the MPTP along with the cardioprotective role of S-nitrosothiols and the potential role for GSNOR

    Compartmentalized Connexin 43 S-Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

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    Objective-To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results-Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NOx concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Conclusion-This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication. (Arterioscler Thromb Vasc Biol. 2011;31:399-407.
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