| Nitration of protein tyrosine residues is commonly observed in the setting of inflammatory, neurodegenerative and autoimmune diseases. It has been proposed that the formation of nitrotyrosine on proteins contributes to disease pathogenesis and the physiological process of aging. To more fully understand the effect of tyrosine nitration in both health and disease, it is important to broadly identify the proteins that undergo nitration under various conditions, as well as the specific tyrosine residues that get modified. To achieve this goal, we have developed a solid-phase proteomic approach for discovery and relative quantification of protein tyrosine nitration sites, and we have used this method to successfully identify 184 nitrated peptides from 107 proteins in peroxynitrite-treated rat brain homogenates.;In addition, we have investigated the validity, mechanism and source of protein denitration, a cellular activity that has been reported to reverse tyrosine nitration in proteins. We have identified nitrated cyclooxygenase-1 as a substrate of this activity and have used its denitration as an assay to successfully validate the authenticity of protein denitration, as well as to further characterize features of protein denitration and to partially purify the enzyme(s) responsible for this activity. The work presented here significantly advances the ability of researchers to study the role of nitrosative stress in human pathology and also increases our understanding of the innate defenses that the body uses to reverse this process. |
