There is strong evidence to implicate nitric oxide (NO), produced from endogenous sources, as a key mediator of toxicity in many important diseases of the nervous system including ischemic stroke, multiple sclerosis, Huntington's disease and Parkinson's disease. The mechanism(s) whereby excessive NO production kills neurons are still unknown, but much evidence points toward involvement of reactive species derived from NO, in particular peroxynitrite, as chief mediators of NO toxicity. Peroxynitrite cannot be measured directly in biological systems, but there is evidence that a newly discovered endogenous post-translational protein modification, protein tyrosine nitration, (PTN) results from the action of peroxynitrite- derived nitronium ion (NO2+) upon tyrosine residues. PIN may be a marker for peroxynitrite. Our first specific aim seeks to establish the presence and localization of PTN within the nervous system of the rat, as well as establish the link between PTN and de novo NO/peroxynitrite production. We will also evaluate the possibility that primary depletion of a critical cytosolic antioxidant, glutathione, can alter PTN. We will use these findings to examine PTN in the 3-nitropropionic acid (3-NP) model of huntington's disease, in which nitric oxide has been very strongly implicated. The second specific aim seeks to establish the structural features of endogenous PTN. Based on our current understanding of the chemical basis of PTN, we hypothesize any sterically available tyrosine residue should be available for nitration. We also seek to examine specific brain proteins that are nitrated, with the intent of developing brain specific markers of PTN. The third specific aim of this proposal is to explore the possibility that PTN itself could have biochemical consequences. There is good evidence that nitrated tyrosine residues are sterically hindered from phosphorylation, and so PTN may be a mechanism whereby NO/peroxynitrite could influence the many signal transduction cascades (including those for several neural growth factors) that utilize tyrosine phosphorylation. This 5-year project seeks to explore biochemical mechanisms by which NO, and reactive species derived from it, affect cells, particularly in the brain. NO has been implicated in a broad range of pathophysiologic processes in the brain, and the potential health relatedness of the studies proposed are therefore broad.
