Protein Tyrosine Nitration and Thiol Oxidation by Peroxynitrite—Strategies to Prevent These Oxidative Modifications
2013, Daiber, Andreas, Daub, Steffen, Bachschmid, Markus, Schildknecht, Stefan, Oelze, Matthias, Steven, Sebastian, Schmidt, Patrick, Megner, Alexandra, Wada, Masayuki, Tanabe, Tadashi, Münzel, Thomas, Bottari, Serge, Ullrich, Volker
The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.
Chemical model systems for cellular nitros(yl)ation reactions
2009-08-15, Daiber, Andreas, Schildknecht, Stefan, Müller, Johanna, Kamuf, Jens, Bachschmid, Markus M., Ullrich, Volker
S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ((.)NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1(-))as an essential intermediate. This requires low cellular fluxes of (.)NO and superoxide (UO2(-)), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine,equal fluxes of (.)NO and UO2(-) were generated and modulated by the addition of (.)NO donors or Cu,Zn superoxide dismutase. As reactants for S-nitros(yl)ation, NADP+-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of (.)NO and UO2(-) at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and Nnitrosation)demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of UNO was as efficient as the 3 (.)NO/1 UO2(-) system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N2O3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described (.)NO/UO2-flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.
Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure
2005-12-30, Daiber, Andreas, Oelze, Matthias, Coldewey, Meike, Kaiser, K., Huth, C., Schildknecht, Stefan, Bachschmid, Markus, Nazirisadeh, Y., Ullrich, Volker, Mülsch, Alexander, Münzel, Thomas, Tsilimingas, N.
The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.
Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes
2016, Mikhed, Yuliya, Bruns, Kai, Schildknecht, Stefan, Jörg, Michael, Dib, Mobin, Oelze, Matthias, Lackner, Karl J., Münzel, Thomas, Ullrich, Volker, Daiber, Andreas
There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products.
By means of HPLC analysis and detection at 380 nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction.
Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite.
Autocatalytic Nitration of Prostaglandin Endoperoxide Synthase-2 by Nitrite Inhibits Prostanoid Formation in Rat Alveolar Macrophages.
2012-11-15, Schildknecht, Stefan, Karreman, Christiaan, Daiber, Andreas, Zhao, Cheng, Hamacher, Jürg, Perlman, David, Jung, Birgit, van der Loo, Bernd, O'Connor, Peter, Leist, Marcel, Ullrich, Volker, Bachschmid, Markus Michael
Prostaglandin endoperoxide H(2) synthase (PGHS) is a well-known target for peroxynitrite-mediated nitration. In several experimental macrophage models, however, the relatively late onset of nitration failed to coincide with the early peak of endogenous peroxynitrite formation. In the present work, we aimed to identify an alternative, peroxynitrite-independent mechanism, responsible for the observed nitration and inactivation of PGHS-2 in an inflammatory cell model. Results: In primary rat alveolar macrophages stimulated with lipopolysaccharide (LPS), PGHS-2 activity was suppressed after 12 h, although the prostaglandin endoperoxide H(2) synthase (PGHS-2) protein was still present. This coincided with a nitration of the enzyme. Coincubation with a nitric oxide synthase-2 (NOS-2) inhibitor preserved PGHS-2 nitration and at the same time restored thromboxane A(2) (TxA(2)) synthesis in the cells. Formation of reactive oxygen species (ROS) was maximal at 4 h and then returned to baseline levels. Nitrite (NO(2)(-)) production occurred later than ROS generation. This rendered generation of peroxynitrite and the nitration of PGHS-2 unlikely. We found that the nitrating agent was formed from NO(2)(-), independent from superoxide ((•)O(2)(-)). Purified PGHS-2 treated with NO(2)(-) was selectively nitrated on the active site Tyr(371), as identified by mass spectrometry (MS). Exposure to peroxynitrite resulted in the nitration not only of Tyr(371), but also of other tyrosines (Tyr). Innovation and Conclusion: The data presented here point to an autocatalytic nitration of PGHS-2 by NO(2)(-), catalyzed by the enzyme's endogenous peroxidase activity and indicate a potential involvement of this mechanism in the termination of prostanoid formation under inflammatory conditions.
Peroxynitrite as regulator of vascular prostanoid synthesis
2009-04-15, Schildknecht, Stefan, Ullrich, Volker
Prostanoids and nitric oxide ((.)NO) are essential modulators of cardiovascular function in health and disease. Among the (.)NO-derived species formed in cells, peroxynitrite (ONOO(-)) is generally associated with its role as nitrating agent under severe pathophysiological conditions. This review, however, highlights a physiological role of peroxynitrite as endogenously formed regulator of prostanoid synthesis in the cardiovascular system. Prostaglandin endoperoxide H2 synthase (PGHS)(1), the central enzyme in the prostanoid pathway was observed to be nitrated and inactivated by high fluxes of peroxynitrite. In contrast, low nanomolar levels, that are formed endogenously in cardiovascular cells, turned out to activate PGHS and therefore prostanoid formation. A further increase in the rates of (.)NO and superoxide ((.)O2(-)) generation, that can be observed after exposure of vascular endothelial cells to endotoxin, results in enhanced levels of peroxynitrite that were shown to selectively nitrate and inactivate prostacyclin (PGI(2))-synthase as one of the dominating terminal prostanoid synthases in the cardiovascular system. As a consequence, accumulation of the intermediate PGH(2) occurs that is capable to activate the thromboxane A(2) (TxA(2)) receptor on the surface of smooth muscle cells to promote vasoconstriction. The nitration of PGI(2)-synthase thus functions as endogenous posttranslational switch that shuts off the PGI(2)-mediated vasodilatory, anti-aggregatory, and anti-adhesive conditions in order to support the transmigration of immune cells from the blood to the sites of an infection. As a third type of interaction between the (.)NO and the prostanoid pathways, an activation of nitrite by the endogenous peroxidase activity of PGHS can lead to an autocatalytic nitration and inactivation of PGHS under conditions of high nitrite and low arachidonic acid levels that mostly prevail in progressive activation stages in cell types that express inducible NOS-2 such as macrophages.
Sensing hypoxia by mitochondria : a unifying hypothesis involving S-nitrosation
2014-01-10, Ullrich, Volker, Schildknecht, Stefan
Sudden hypoxia requires a rapid response in tissues with high energy demand. Mitochondria are rapid sensors for a lack of oxygen, but no consistent mechanism for the sensing process and the subsequent counter-regulation has been described.
Unique features of the low molecular weight probe salicylaldehyde in the detection of nanomolar peroxynitrite fluxes
2012, Mikhed, Yuliya, Bruns, Kai, Jörg, Michael, Kerahrodi, Jasmin Ghaemi, Gottschlich, Anna, Oelze, Matthias, Schildknecht, Stefan, Ullrich, Volker, Daiber, Andreas
Autocatalytic tyrosine nitration of prostaglandin endoperoxide synthase-2 in LPS-stimulated RAW 264.7 macrophages
2006-02-03, Schildknecht, Stefan, Heinz, Kathrin, Daiber, Andreas, Hamacher, Jürg, Kavaklí, Cengiz, Ullrich, Volker, Bachschmid, Markus
In the literature, biological tyrosine nitrations have been reported to depend not only on peroxynitrite but also on nitrite/hydrogen peroxide linked to catalysis by myeloperoxidase. In endotoxin-stimulated RAW 264.7 macrophages, we have detected a major nitrotyrosine positive protein band around 72 kDa and identified it as prostaglandin endoperoxide synthase-2 (PGHS-2). Isolated PGHS-2 in absence of its substrate arachidonate was not only tyrosine-nitrated with peroxynitrite, but also with nitrite/hydrogen peroxide in complete absence of myeloperoxidase. Our data favor an autocatalytic activation of nitrite by PGHS-2 with a subsequent nitration of the essential tyrosine residue in the cyclooxygenase domain. Under inflammatory conditions, nitrite formed via NO-synthase-2 may therefore act as an endogenous regulator for PGHS-2 in stimulated macrophages. Nitration of PGHS-2 by the autocatalytic activation of nitrite further depends on the intracellular concentration of arachidonate since arachidonate reacted competitively with nitrite and could prevent PGHS-2 from nitration when excessively present.