The objective of the proposed research is to identify and characterize the biochemical events that mediate the effects of nitric oxide (NO) in neurons. NO has several roles in the CNS including regulating neuronal morphology, growth, plasticity, and survival. An emerging concept is that these effects are mediated by the covalent modification of proteins by NO on cysteine residues. This modification, S-nitrosylation, is difficult to detect using currently available approaches. In recent work, we have developed methods that allow S-nitrosylated proteins to be detected by standard Western blotting. Further, using a proteomic approach, we have identified large numbers of S-nitrosylated cytosolic proteins using a combination of protein purification by streptavidin-affinity chromatography and high-throughput mass spectrometry (MS). As part of our overall goal to understand the role of NO in the brain and to understand the mechanisms by which NO mediates its effects in neurons, the specific aims of this proposal are: (1) To identify endogenously S-nitrosylated proteins. Experiments in Aim I describe modifications of our novel methodology which will result in the purification, characterization, and identification of S-nitrosylated proteins that are found in synaptic densities, neuronal membranes, and other neuronal compartments enriched in signaling proteins and NO synthesis. (2) To develop methods to characterize the stoichiometry and sites of S-nitrosylation in proteins and identify consensus sites for S-nitrosylation in proteins. Experiments in Aim II describe novel MS approaches to identify sites of S-nitrosylation of proteins. Further, we describe a novel technique, """"""""nitrosopeptide mapping"""""""" which allows numerous S-nitrosylation events in one protein to be distinguished at once. Using these techniques we describe experiments that will provide insight into consensus and other chemical requirements for Snitrosylation. (3) To characterize S-nitrosylation events that occur following neurotransmitter stimulation. In this Aim, we describe experiments to detect and identify proteins that are S-nitrosylated following neurotransmitter stimulation of cultured neurons. We will also characterize mechanisms that lead to the denitrosylation of these proteins. Together, the experiments described in this proposal will systematically analyze neuronal compartments for S-nitrosylated proteins and will provide insight into the role of Snitrosylation in neuronal signal transduction. Further, these experiments will provide general methods that will facilitate the analysis of S-nitrosylation in a variety of biological systems.
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