Selective nitration of histone tyrosine residues in vivo in mutatect tumors

Nitric oxide-derived reactive species have been implicated in many disorders. Protein nitrotyrosine is often used as a stable marker of these reactive species. Using immunohistochemistry, we have previously detected nitrotyrosine in murine Mutatect tumors, where neutrophils are the principal source of nitric oxide. We now report on the identification of several prominent nitrotyrosine-containing proteins. Using Western blot analysis, nitrotyrosine in higher molecular mass proteins (>20 kDa) was detected in tumors containing a high number of neutrophils but not in tumors with fewer neutrophils. Staining for nitrotyrosine was consistently seen in low molecular mass proteins (less than or equal to15 kDa), regardless of the level of neutrophils. Protein nitrotyrosine was not seen in Mutatect cells growing in vitro. Treatment with nitric oxide donors produced nitration of less than or equal to15-kDa proteins, but only after extended periods. These small proteins, both from tumors and cultured cells, were identified by mass spectrometry to be his-tones. Only a subset of tyrosine residues was nitrated. Selective nitration may reflect differential accessibility of different tyrosine residues and the influence of neighboring residues within the nucleosome. The prominence of histone nitration may reflect its relative stability, making this post-translational modification a potentially useful marker of extended exposure of cells or tissues to nitric oxide-derived reactive species.