Oxidation of lipoproteins is important for the initiation and propagation of the atherosclerotic lesion and may involve secondary oxidants derived from nitric oxide. Nitric oxide (NO) reacts at near diffusion limited rates with superoxide (O2-.) to form the strong oxidant, peroxynitrite (ONOO-). Nitration on the ortho position of tyrosine is a major product of peroxynitrite attack on proteins. Nitrotyrosine was detected in atherosclerotic lesions of formalin-fixed human coronary arteries with polyclonal and monoclonal antibodies. Binding was pronounced in and around foamy macrophages within the atheroma deposits. Nitration was also observed in early subintimal fatty streaks. Antibody binding was completely blocked by co-incubation with 10mM nitrotyrosine, but not by equivalent concentrations of aminotyrosine or phosphotyrosine. The presence of nitrotyrosine indicates that oxidants derived from nitric oxide such as peroxynitrite are generated in human atherosclerosis and may be involved in its pathogenesis.

NO 2 Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO 2 Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO 2 Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of ␣-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxylterminal tyrosination͞detyrosination cycle of ␣-tubulin, incorporation of NO 2 Tyr shows apparent irreversibility. Nitrotyrosination of ␣-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelialbarrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of ␣-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of ␣-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motorand microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO 2 Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the ␣-tubulin tyrosination͞detyrosination cycle plays in microtubule function. Nitric oxide (• NO) is a pervasive signaling molecule generated from L-arginine via the catalytic action of both constitutive and inducible forms of • NO synthases (1). A large body of evidence has amassed in the last decade, establishing the operative role of inducible • NO synthase in the pathogenesis of inflammatory, infectious, and degenerative human diseases (2). The detrimental effects ascribed to • NO often arise from its conversion to more reactive species through reactions with partially reduced oxygen species (3).The pathophysiological actions of• NO congeners are primarily rooted in their capacity to alter the function of biological macromolecules through covalent modifications. A metabolite generally reflecting in vivo production of reactive nitrogen intermediates is the amino acid derivative 3-nitrotyrosine (NO 2 Tyr). Evidence for NO 2 Tyr formation in vivo was found when the free amino acid and its deaminated͞ decarboxylated metabolite 3-nitro-4-hydroxyphenylacetic acid were detected as excretory products in human urine (4). The significance of NO 2 Tyr in vivo is highlighted further by observations that protein-linked NO 2 Tyr is markedly elevated in a broad range of human diseases and clinical disorders (5). In vitro studies have identifi...

No abstract

Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO), which may be the damaging gain-offunction resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in a-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SODcatalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.

No abstract

No abstract

No abstract

Nitrotyrosine is an important marker for the formation of peroxynitrite and possibly other reactive nitrogen species derived from nitric oxide in vivo (1). Pathological conditions can substantially increase the production of nitric oxide, yet this molecule itself does not generally yield nitration of tyrosine residues in proteins when added to biological samples (1,2). However nitric oxide reacts at near diffusion-limited rates with superoxide (O(2) (-)) to form the strong oxidant peroxynitrite (ONOO(-)) (3). Nitration on the 3-position of tyrosine is a major product of peroxynitrite attack on proteins (4,5). Certainly, small amounts of nitrotyrosine can be produced in vivo by other mechanisms (6), but peroxynitrite is by far the most efficient mechanism for nitrating tyrosine under biologically relevant conditions with natural antioxidants and alternative targets present.