Selective Nitration of Histone Tyrosine Residues in Vivo in Mutatect Tumors

Abstract

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 (<15 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 <15-kDa proteins, but only after extended periods. These small proteins, both from tumors and cultured cells, were identified by mass spectrometry to be histones. 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.Nitric oxide (NO ⅐ ) 1 -derived reactive nitrogen oxide species (RNOS) are cytotoxic and mutagenic, and have been implicated in the pathogenesis of several inflammatory disorders (1-9). Inflammatory cells, such as macrophages, monocytes, and neutrophils produce a relatively large amount of RNOS. These reactive species may exert deleterious effects by modifying or damaging various cellular targets including DNA, lipids, and proteins (10 -12). One of the targets in proteins is tyrosine (Tyr), which can be converted into a fairly stable end-product, 3-nitrotyrosine (NTyr) (13). The presence of free NTyr amino acids or NTyr-containing proteins in biological samples is used as a molecular marker of RNOS production in a tissue (13). In addition, nitration of Tyr residues in proteins may alter protein function (4, 14 -17), which may have both physiological and pathological significance. The presence of protein NTyr has been reported in inflammatory disorders such as Helicobacter pylori infection, Crohn's disease, ulcerative colitis, Wegener's granulomatosis, cystic fibrosis, asthma, obliterative bronchiolitis, and rheumatoid arthritis (2-9). The level of protein NTyr has been found to correlate with the severity of inflammatory diseases (18 -20). Protein NTyr has also been detected in some tumors (21-24). A high level of protein NTyr correlates with poor outcome in melanoma patients (25). Although a high level of protein NTyr has been detected in vivo in human or animal diseases, only a limited number of NTyr-containing proteins has been identified (26 -34).Several methods have been employed to...

Expression of Inducible Nitric-oxide Synthase and Intracellular Protein Tyrosine Nitration in Vascular Smooth Muscle Cells

“…Localization of nitrated proteins within outer mitochondrial membranes has also been observed in dendrites in rat brain (44). The localization of nitrated proteins in the nucleus observed in this model is consistent with the recent demonstration of nitration of Tyr 98 in histone 4 and Tyr 42 in histone 2B in tumors (45). Considering that the intracellular compartment is densely populated by proteins, the localization of nitrated proteins in specific cellular compartments implies that specific proteins are targeted for nitration.…”

Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins

“…Some reports describe chemical nitration of histones (30)(31)(32), whereas others demonstrate that nuclear staining for nitrotyrosine by immunohistochemistry is often observed in tissues and in NO-exposed cell cultures (33)(34)(35). Recently, Haqqani et al (36) described that histones are the most prominent nitrated proteins in the Mutatect tumor tissue as well as in the cultured Mutatect cells that are exposed to NO. They also suggest tyrosine nitration of Histone H1 in vivo, which support the view that Histone H1.2 might be an intrinsic substrate for denitrase.…”

Nitrosative protein tyrosine modifications: biochemistry and functional significance

“…Among the nitrated proteins found in cell nuclei, p53 (72), histone (73), and Cu, Zn-SOD (74) regulate apoptosis and tumor growth. Many of the cytosolic proteins with nitrated tyrosine residues are also implicated in diseases such as diabetes, inflammation, and neurodegenerative diseases.…”