Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

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Reactive oxygen species (ROS) such as superoxide radical anion (O2•¯), hydroxylradical (OH•), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) that are produced during the metabolism of oxygen under oxidative stress in aerobic organisms destroy several key biomolecules and lead to a number of disease states. Mammalian systems possess several effective defense mechanisms including antioxidant enzymes to detoxify these ROS. The selenocysteine-containing Glutathione peroxidase (GPx) is particularly an efficient enzyme in the detoxification of H2O2 and other hydroperoxides by using glutathione (GSH) as cofactor. The chemistry at the active siteof GPx has been extensively investigated with the help of synthetic selenium compounds. Although the anti-inflammatory compound ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one) is undergoing phase III clinical trial as antioxidant, the chemistry of ebselen is still not understood. The present study on a number of ebselen derivatives with various N-substitutions reveals that the substitution at the N atom is important for the antioxidant activity. This study also suggests that the nature for thiol cofactor has a dramatic effect on the GPx activity of ebselen derivatives. It has been shown that ebselen exhibits very poor catalytic activity in the presence of aromatic thiols mainly due to strong Se….O nonbonded interactions that lead to extensive thiol exchange reactions in the selenenyl sulfide intermediate. To prevent the se….O interactions, a series of tertiary amide-based diselenides have been synthesized along with their secondary amide counterparts. Detailed structure-activity correlation studies reveal that the GPx-like activity of the sec-amide-based compounds can be significantly enhanced by the substitution at the free-NH group of sec-amide functionality. The N,N-dialkylbenzylamine-based diselenides exhibit their catalytic activities via the generation of selenols which was confirmed by the reaction with anti-arthritic gold(I) compounds. Interestingly, the replacement of the hydrogen atom at the 6th position of the benzene ring of N,N-dialkylbenzylamine-based diselenides by a methoxy group prevents the thiol exchange reactions mainly be weakening the Se…N interactions and thus enhances the GPx activity. On the other hand, the catalytic activity of the tert-amine-based diselenides can also be increased by replacing the tert-amino groups with the corresponding sec-amine moieties. It has been observed that the basic amino group in the amine-based diselenides deprotonates the selenol and also the thiol cofactor, which is crucial for the higher catalytic activities of the amine-based compounds. Peroxynitrite (PN, ONOO), a strong nitrating agent, is known to inactivate a number of proteins, enzymes and other biomolecules by nitration of tyrosine residues. In this study, we have shown that the commonly used antithyroid drugs and their analogues inhibit protein tyrosine nitration. This study reveals that antithyroid agents having PN scavenging activity may be beneficial of hyperthyroidism as these compounds may protect the thyroid gland from nitrative or nitrosative stress.