Site-specific Nitration Research Articles

Site-Specific Nitration Differentially Influences τ Assembly in Vitro

Previously, we reported that the microtubule-associated tau protein, the major constituent of neurofibrillary tangles (NFTs) in Alzheimer's brain, undergoes site-selective nitration by peroxynitrite (ONOO-) and that this event inhibits tau polymerization in vitro [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we extend our analysis of tau nitration to include mutant tau proteins singly nitrated at each residue targeted by ONOO- in vitro (Tyr18, Tyr29, Tyr197, and Tyr394). Using our polymerization paradigm, we demonstrate that site-specific Tyr nitration differentially alters the rate and/or extent of tau assembly and generates robust changes in filament morphology. As determined by quantitative electron microscopy, select nitration of residues Tyr29 and Tyr197 increases the average length of synthetic tau filaments but does not alter the steady-state polymer mass. In contrast, site-specific nitration of residues Tyr18 and Tyr394 decreases the average length and/or number of synthetic filaments, resulting in a significant reduction in filamentous mass and an increase in tau critical concentration. Intriguingly, affinity measurements demonstrate that nitrative modifications do not preclude formation of the Alz-50 epitope, a pathological tau conformation detectable in authentic paired helical filaments (PHFtau). In fact, the Alz-50 antibody binds filaments assembled from nitrated mutant tau with higher avidity than wild-type filaments, even in instances where the overall filamentous mass is reduced. Taken together, our results suggest that site-specific nitration modulates the nucleation and/or elongation capacity of assembly-competent tau and that assumption of the Alz-50 conformation may be necessary, but not sufficient, to induce filament formation.

Site-Specific Nitration and Oxidative Dityrosine Bridging of the τ Protein by Peroxynitrite: Implications for Alzheimer's Disease

Alzheimer's disease (AD) is a progressive amnestic disorder typified by the pathological misfolding and deposition of the microtubule-associated tau protein into neurofibrillary tangles (NFTs). While numerous post-translational modifications influence NFT formation, the molecular mechanisms responsible for tau aggregation remain enigmatic. Since nitrative and oxidative injury have previously been shown to play a mechanistic role in neurodegeneration, we examined whether these events influence tau aggregation. In this report, we characterize the effects of peroxynitrite (ONOO-)-mediated nitration and oxidation on tau polymerization in vitro. Treatment of tau with ONOO- results in 3-nitrotyrosine (3-NT) immunoreactivity and the formation of heat-stable, SDS-insoluble oligomers. Using ESI-MS and HPLC with fluorescent detection, we show that these higher-order aggregates contain 3,3'-dityrosine (3,3'-DT). Tyrosine (Tyr) residues are critical for ONOO(-)-mediated oligomerization, as tau proteins lacking all Tyr residues fail to generate oligomers upon ONOO- treatment. Further, tau nitration targets residues Y18, Y29, and to a lesser degree Y197 and Y394, and nitration at these sites inhibits in vitro polymerization. The inhibitory effect of nitration on tau polymerization is specific for the 3-NT modification, as pseudophosphorylation at these same Tyr residues does not inhibit tau assembly. Our results suggest that the nitrative and oxidative roles of ONOO- differentially affect tau polymerization and that ONOO(-)-mediated cross-linking could facilitate tau aggregation in AD.

Nitric oxide, oxidants, and protein tyrosine nitration.

The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of (.)NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO(-)) and nitrogen dioxide ((.)NO2), formed as secondary products of (.)NO metabolism in the presence of oxidants including superoxide radicals (O2(.-)), hydrogen peroxide (H2O2), and transition metal centers. The precise interplay between (.)NO and oxidants and the identification of the proximal intermediate(s) responsible for nitration in vivo have been under controversy. Despite the capacity of peroxynitrite to mediate tyrosine nitration in vitro, its role on nitration in vivo has been questioned, and alternative pathways, including the nitrite/H2O2/hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitration in vivo, and (ii) most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (CO3(.-)) and/or oxo-metal complexes oxidizing tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with (.)NO2 to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of (.)NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.