The nitration of tau protein in neurone-like PC12 cells

Tyrosine nitration of proteins is emerging as a post-translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor-induced neuronal differentiation, we now find that nitration occurs on the microtubule-associated protein tau. In differentiated PC12 cells, we have identified as tau a nitrated protein that co-immunoprecipitates with alpha-tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of tau occurs in a physiological process and suggests that it could play a role in neuronal differentiation.     

NO-dependent protein nitration: a cell signaling event or an oxidative inflammatory response?

Tyrosine nitration is becoming increasingly recognized as a prevalent, functionally significant post-translational protein modification that serves as an indicator of nitric oxide (√NO)-mediated oxidative inflammatory reactions. Nitration of proteins modulates catalytic activity, cell signaling and cytoskeletal organization. Several reactions mediate protein nitration, and all predominantly depend on √NO- and nitrite-dependent formation of nitrogen dioxide, a species capable of nitrating aromatic amino acids, nucleotides and unsaturated fatty acids. Here, we review the mechanisms that mediate in vivo protein nitration and how nitration of specific tyrosine residues impacts on protein function.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Protein tyrosine nitration in the cell cycle

Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.     

Nitration and inactivation of tyrosine hydroxylase by peroxynitrite

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.     

Proteolytic degradation of tyrosine nitrated proteins

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.     

Detection of in vivo protein tyrosine nitration in petite mutant of Saccharomyces cerevisiae: Consequence of its formation and significance

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with physiological and pathophysiological conditions. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group. In our previous study we first time showed that PTN occurs in vivo in Saccharomyces cerevisiae. In the present study we observed occurrence of PTN in petite mutant of S. cerevisiae which indicated that PTN is not absolutely dependent on functional mitochondria. Nitration of proteins in S. cerevisiae was also first time confirmed in immunohistochemical study using spheroplasts. Using proteosomal mutants Rpn10Δ, Pre9Δ, we first time showed that the fate of protein nitration in S. cerevisiae was not dependent on proteosomal clearing and probably played vital role in modulating signaling cascades. From our study it is evident that protein tyrosine nitration is a normal physiological event of S. cerevisiae.     

Temporal patterns of tyrosine nitration in embryo heart development

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.     

Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry

Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.     

Site-specific nitration and oxidative dityrosine bridging of the tau 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.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

Protein tyrosine nitration in the mitochondria from diabetic mouse heart. Implications to dysfunctional mitochondria in diabetes

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.     

Characterization of Nitrotyrosine-Modified Proteins in Cerebrospinal Fluid

Background

HIV-associated neurocognitive disorders (HAND) has been associated with the up-regulation of various oxidative stress pathways. Previous studies have linked the neuronal damage observed in individuals diagnosed with HAND to increased nitrotyrosine modification of neuronal proteins.

Materials and methods

Tyrosine nitration alters protein structure and function, affects biological half-life, and potentially prevents the phosphorylation of key tyrosine residues involved in signal transduction pathways. Therefore, in this study we employed proteomics-based experimental approaches to investigate nitrotyrosine-modified proteins in pooled cerebrospinal fluid (CSF) of individuals diagnosed with HAND. To identify specific nitrotyrosine-modified proteins in the CSF of individuals diagnosed with HAND, affinity purification and high-performance tandem mass spectrometry are utilized in a “bottom-up” proteomics approach.

Results

From tandem mass spectrometric analysis, we identified major proteins that underwent nitration as a result of nitro-oxidative stress in the CSF of individuals diagnosed with HAND. We also utilized analytical and biochemical techniques to characterize the expression and modification site of in vivo nitrated lipocalin-type prostaglandin-D synthase in HAND CSF.     

Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration

Tyrosine nitration is a common modification to proteins in vivo, but the reactive nitrogen species responsible for nitration are often studied in vitro using just the amino acid tyrosine in simple phosphate solutions. To investigate which reactive nitrogen species could nitrate proteins in a complex biological system, we exposed rat heart and brain homogenates to peroxynitrite, nitric oxide under aerobic conditions, and other putative nitrating agents. Peroxynitrite was by far the most efficient nitrating agent when alternative targets were available to compete with tyrosine. Curiously, proteins in heart homogenates were substantially more resistant to nitration than brain homogenates. Ultrafiltration to remove low molecular weight compounds made the heart proteins equally susceptible as the brain proteins to nitration. Endogenous ascorbate and free thiols had little effect on nitration by peroxynitrite in either heart or brain. However, accumulation of urate formed by the oxidation of hypoxanthine by xanthine dehydrogenase and oxidase in heart appeared to be the major inhibitor of nitration. Heart homogenates treated with uricase, which converts urate to allantoin, showed equivalent nitration as in brain homogenates. Urate, as assayed by HPLC, was 58 +/- 8 microM in heart but only 4 +/- 2 microM in brain homogenates. Although xanthine dehydrogenase conversion to a free radical-producing oxidase can serve as an important source of superoxide and hydrogen peroxide during ischemia/reperfusion, our results suggest that urate formation by xanthine dehydrogenase may provide a significant antioxidant defense against peroxynitrite and related nitric oxide-derived oxidants.     

Mitochondrial tyrosine nitration precedes chronic allograft nephropathy

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2–52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.     

A stringent approach to improve the quality of nitrotyrosine peptide identifications

Tyrosine nitration is the consequence of a complex machinery of formation and merging of oxygen and nitrogen radicals, and has been associated with both physiological pathways as well as with several human diseases. The latter turned this posttranslational protein modification into an interesting biomarker, being either a consequence of the disease or a factor contributing to the disease onset. However, the interpretation of MS and MS/MS data of peptides containing nitrotyrosine has proven to be very challenging and consequently, the risk of linking MS/MS spectra to incorrect peptide sequences exists and has been reported. Here, we discuss the causes of data misinterpretation and describe a general method to avoid mistakes of MS/MS spectrum misinterpretation. Central in our approach is the reduction of nitrotyrosine into aminotyrosine and the use of the Peptizer algorithm to inspect MS/MS quality-related assumptions.     

铁过载促进小鼠肝组织发生蛋白质酪氨酸硝化

蛋白质酪氨酸硝化是一种蛋白质翻译后的修饰,其存在会影响酶的催化活性,细胞信号转导和细胞骨架结构．在铁过载情况下,存在引起蛋白质酪氨酸硝化的有利环境,但目前尚无实验证实．本文运用腹腔注射右旋糖苷铁造成小鼠铁过载模型,通过免疫印迹法发现,在铁过载情况下,肝中诱导型一氧化氮合酶表达显著高于正常对照小鼠;铁过载小鼠肝中总体蛋白质硝化程度高于正常小鼠;铁过载引起的蛋白质酪氨酸硝化有一定的选择性,在铁过载小鼠肝中发现一些新的被硝化蛋白质条带（约 57 kD、 35 kD）．上述结果证实,铁过载会促进肝蛋白质酪氨酸硝化．     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.