Molecular and cellular mechanisms of tyrosine nitration

Fascinating research of the last decade in areas as diverse as human memory, penile erection and immune response has revealed an important transduction mechanism in which nitric oxide (NO) acts as a cellular messenger. Besides its physiological functions, NO causes tissue damage in the course of diseases associated with a systemic overproduction of NO. One of the. mechanisms by which excess NO can injure tissues is by its rapid reaction with superoxide anion (O2 .- ) to give peroxynitrite, a potent oxidant thought to be a key mediator of NO toxicity in atherosclerosis, cardiac ischemia/reperfusion injury, stroke, and other diseases involving inflammatory oxidative stress. In vitro, peroxynitrite has been shown to react with virtually all classes of biomolecules. The reaction of synthetic peroxynitrite with free or protein-bound tyrosine results in the formation of 3-nitrotyrosine. Based on the detection of 3-nitrotyrosine in injured tissues, peroxynitrite formation has been suggested to be the major cause for NO toxicity. However, mechanisms of tyrosine nitration unrelated to peroxynitrite formation have been described, and we found that peroxynitrite generated from NO and O2 .- does not nitrate free tyrosine in vitro. It is the aim of this project to study the molecular and cellular mechanisms contributing to tyrosine nitration in inflammation and cardiac ischemia-reperfusion injury. So far, tyrosine nitration has been extensively studied with synthetic peroxynitrite, an experimental approach that does not reflect in vivo situations in which peroxynitrite is formed from NO and O2 .- continuously generated from different sources. To account for this apparent lack of knowledge, the first part of the proposal encompasses studies on tyrosine nitration in isolated and cultured cells in which the production of NO and O2 .- will be turned on independently by addition of selective stimuli of the respective pathways, followed by analyses of relevant biochemical parameters, such as 3-nitrotyrosine levels, release rates of NO, O2 .- and peroxynitrite, and NO synthase activity. The second part of the proposal focuses on the functional consequences of tyrosine nitration in an experimental model of cardiac ischemia/reperfusion injury. Hearts isolated from genetically engineered mice lacking or overexpressing enzymes thought to be key players in tyrosine nitration (NO synthase, superoxide dismutase, myeloperoxidase) will be subjected to global ischemia and tested for tyrosine nitration after increasing periods of reperfusion. The results will be compared with functional parameters of heart function (left ventricular pressure, cardiac output, coronary flow) to test for a possible causal relationship between tyrosine nitration and impaired cardiac function. In summary, the proposed studies are expected to elucidate the biosynthetic pathways accounting for the well documented occurrence of 3-nitrotyrosine in vivo and to provide new therapeutic strategies for the effective treatment of diseases associated with inflammatory oxidative stress.

Nitric oxide (NO) is a widespread biological signal molecule, which is involved, in biological processes as diverse as regulation of blood pressure, platelet aggregation, neurotransmission in the brain, penile erection, and immune defense against invading pathogens. Besides these important physiological functions, overproduction of NO may be deleterious and contribute to tissue injury in various inflammatory, infectious, and neurodegenerative diseases. Unlike other cellular messenger molecules, which act via specific interactions with ligand binding sites of target protein (enzymes, receptors, membrane transporters), nitric oxide (NO) exerts its effects through chemical reactions with cellular constituents, rendering the biological chemistry of NO important for the understanding of NO physiology and pathophysiology. Formation of peroxynitrite, a reactive molecule that exerts deleterious effects through modification of cellular proteins, is one major toxic reaction of NO. In this project we studied the mechanism and biological significance of cellular protein tyrosine nitration. We were able to demonstrate that peroxynitrite does not trigger significant tyrosine nitration at low steady-state concentrations as found upon in situ generation in cells. Based on these results we screened for alternative nitration reactions in cells, in particular in a murine macrophage cell line activated by cytokines. In this model we observed that protein tyrosine nitration was blocked by inhibitors of the enzyme myeloperoxidase but not by peroxynitrite scavengers. In addition, we observed that nitration coincided with the formation of nitrite, the substrate for myeloperoxidase-catalyzed nitration, but not with peroxynitrite generation. These results indicated that protein tyrosine nitration in cells is not catalyzed by peroxynitrite but due to myeloperoxidase-catalyzed oxidation of nitrite. Based on these results we studied protein tyrosine nitration in isolated peritoneal macrophages from mice. The cells were either activated in vitro by cytokines or obtained from animals that had been subjected to systemic inflammation by treatment with the bacterium C. parvum. In both the in vitro and the in vivo model peroxynitrite formation was almost completely inhibited by the established peroxynitrite scavenger MnTBAP; however, tyrosine nitration was hardly affected by this compound. As observed with the macrophage cell line, tyrosine nitration was markedly delayed compared to peroxynitrite formation in freshly isolated peritoneal macrophages. Taken together, our results show that peroxynitrite does not trigger nitration of cellular proteins neither in vitro nor in vivo and indicate that this reaction is caused by myeloperoxidase-catalyzed oxidation of nitrite to nitrogen dioxide radicals. Thus, potent and selective myeloperoxidase inhibitors and/or nitrogen dioxide scavengers could be therapeutically useful for the treatment of various infectious inflammatory diseases associated with increased oxidative stress.