Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.     

Modifications of glyceraldehyde-3-phosphate dehydrogenase induced by increasing concentrations of peroxynitrite: early recognition by 20S proteasome

Peroxynitrite, a potent oxidizing and nitrating species, induces covalent modifications of biomolecules in a number of pathological conditions. In previous studies with S. cerevisiae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as being especially susceptible to nitration by peroxynitrite. The activity of this enzyme was strongly inhibited by low doses of peroxynitrite in yeast and in cultured rat astrocytes. Here, the sequence of modifications of isolated mammalian GAPDH induced by increasing concentrations of peroxynitrite is demonstrated to be as follows: (i) oxidation, leading to inactivation and to enhanced susceptibility of GAPDH for proteasomal degradation, (ii) oligomer formation, and (iii) nitration. In our study the susceptibility for degradation by isolated 20S proteasome was by far the most sensitive parameter for peroxynitrite-induced damage to GAPDH, implying that this might also occur under pathological conditions where peroxynitrite is generated at low concentrations in vivo.     

Peroxynitrite and protein tyrosine nitration of prostacyclin synthase

When working on the regulation of prostacyclin synthase (PGIS), we found that PGIS was selectively inhibited by peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anion and nitric oxide (NO) at a rate of diffusion-controlled. None of the cellular antioxidants studied (i.e. GSH, Vitamins C and E, and others) prevented the inhibition of ONOO- on PGIS. This unexpected behavior was explained by a catalytic reaction of the iron-thiolate center of PGIS with ONOO- anion. In contrast, ONOO- activated both thromboxane A2-synthase and cyclooxygenases. In addition, we demonstrated that sub-micromolar levels of ONOO- inhibited PGI2-dependent vasorelaxation and triggered a PGH2-dependent vasospasm, indicating that ONOO- increased PGH2 formation as a consequence of PGIS nitration. We have subsequently demonstrated that endogenous ONOO- caused PGIS nitration and TxA2 activation in several diseased conditions such as atherosclerotic vessels, hypoxia-reperfusion injury, cytokines-treated cells, diabetes, as well as hypertension. Since NO is produced physiologically it seems that excessive formation of superoxide not only eliminates the vasodilatory, growth-inhibiting, anti-thrombotic and anti-adhesive effects of NO and PGI2 but also allows and promotes an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2. We conclude that the nitration of PGIS nitration might be a new pathogenic mechanism for superoxide-induced endothelium dysfunction often observed in vascular diseases such as atherosclerosis, hypertension, ischemia, endotoxic shock, and diabetes.     

Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO- may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO- on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO- or decomposed ONOO- in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2'-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO- treatment in vitro inhibited enterocyte proliferation. ONOO- treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO- blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO- may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.     

S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO-), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O2- that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO- detoxification activity of PrxII E, causing a dramatic increase of ONOO--dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO-, indicating that the PrxII E modulation of ONOO- bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species- and NO-mediated signaling events.     

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.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.     

Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.     

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.     

Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite

The protective effects of resveratrol (3, 4', 5-trihydroxystilbene; present naturally in different plants) against the oxidative/nitrative damage of human plasma proteins induced by peroxynitrite (ONOO-) were studied and compared with those of deferoxamine (DFO; a natural siderophore isolated from Streptomyces pilosus), which is a typical and well-known antioxidant. We also studied the effect of ONOO- on plasma lipid peroxidation and the role of tested antioxidants in this process. ONOO- at the used concentrations (0.01-1 mM) showed toxicity to human plasma components. Exposure of plasma to ONOO- (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins (approximately 4-fold and 76-fold, respectively) and in a distinct augmentation of lipid peroxidation (approximately 2-fold). In the presence of 0.1-mM resveratrol, a distinct decrease of carbonyl group formation and tyrosine nitration in plasma proteins caused by 0.1-mM ONOO- was observed (by approximately 70% and 65%, respectively). Addition of 0.1-mM DFO to plasma also distinctly reduced the level of carbonyl groups and nitrotyrosines caused by 0.1-mM ONOO- (by approximately 50% and 60%, respectively). Moreover, these antioxidants also inhibited plasma lipid peroxidation induced by ONOO- (0.1 mM). The obtained results indicate that in vitro resveratrol, like well-known antioxidant DFO, has inhibitory effects on ONOO- -mediated oxidation of proteins and lipids in human plasma.     

Peroxynitrite-mediated tau modifications stabilize preformed filaments and destabilize microtubules through distinct mechanisms

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated tau protein that promote its pathological self-assembly and displacement from the MT lattice. Previously, we showed that peroxynitrite (ONOO-) induces the oxidative 3,3'-dityrosine (3,3'-DT) cross-linking and site-selective nitration of tau monomers [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we examined the effects of ONOO(-)-mediated modifications on two key elements of tau pathobiology: (1) the stability of preformed tau filaments and (2) the ability of monomeric tau to promote tubulin assembly. Here, we report that treatment of synthetic tau filaments with ONOO- generates heat-stable, SDS-insoluble aggregates with a significantly reduced mobility by SDS-PAGE compared to that of nontreated filaments. Ultrastructurally, these aggregates appear to be cross-linked via interfilament bridges. Using LC-MS/MS and HPLC with fluorescent detection, we demonstrate that covalent 3,3'-DT linkages are present within these higher-order aggregates. Similar to monomeric tau, filamentous tau exhibits a hierarchical pattern of nitration following ONOO- treatment with site selectivity toward the amino-terminal residues Tyr18 and Tyr29. Further, select nitration of residues Tyr18, Tyr29, Tyr197, and Tyr394, events known to stabilize the pathological Alz-50 conformation [Reynolds et al. (2005) Biochemistry 44, 13997-14009], inhibits the ability of monomeric tau to promote tubulin assembly. This effect is specific for the 3-NT modification, as mutant tau proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs. Collectively, our results suggest that ONOO(-)-mediated modifications stabilize tau filaments via 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers. Moreover, assumption of the Alz-50 conformation may be the mechanism through which tau nitration modulates MT stability.     

Relationship between protein nitration and oxidation and development of hyperoxic seizures.

Recent studies have implicated nitric oxide (NO*) as a mediator of CNS hyperbaric O2 (HBO2) toxicity. One mechanism by which NO* may contribute to HBO2-induced brain toxicity involves a neurotoxic, pro-oxidative action of NO* via the formation of the potent oxidant peroxynitrite (ONOO-). The present study compares: (a) the formation of protein nitrotyrosine as a marker of ONOO- accumulation and (b) protein oxidation as an indicator of reactive oxygen species production during HBO2 exposure. Rats were exposed to 5 atm 100% O2 to pre-convulsive exposure or until the occurrence of electroencephalographic (EEG) seizures. After exposures, brains were analyzed for protein nitrotyrosine (NT) and protein carbonyl measurement by Western blot and for superoxide dismutase (SOD) activity by NBT assay. The results show a significant increase in protein NT, exceeding control level by several fold. There was only a slow and non-significant increase in the quantity of oxidized proteins during the pre-convulsive phase of HBO2 exposure. Levels of both protein NT and protein carbonyls were significantly (p<0.05) elevated after seizures. Total SOD activity was not changed during preconvulsive exposures, but was significantly (p<0.05) elevated post-seizures. The specific neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), significantly reduced the increases in seizure-induced protein NT and protein carbonyl and at the same time very effectively (p<0.05) delayed onset of HBO2 seizures. Pre-seizure increases in protein NT might indicate its role in the mechanism of HBO2-induced brain toxicity. This is supported by the observed capacity of 7-NI to inhibit tyrosine nitration and increase time to seizure.     

Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.     

Amyloid beta modulated the selectivity of heme-catalyzed protein tyrosine nitration: an alternative mechanism for selective protein nitration

Protein tyrosine nitration is a post-translational modification associated with numerous pathological conditions. The biological consequences of this modification strongly depend on the site selectivity. Unfortunately, to date there is still no reliable model for predicting the selectivity of protein tyrosine nitration. Previously, we found that amyloid beta (Aβ) changed the selectivity of enolase tyrosine nitration upon binding to heme. It seemed that there was a link between the hydrophilicity of Aβ and the site-specific tyrosine nitration. We further investigated the role of the hydrophilicity of the molecules that bind to heme in the selectivity of protein tyrosine nitration. We found that Aβ(1-16), Aβ(1-20), and Aβ(1-40), upon binding to heme and interacting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a site-specific manner, differently modulated the site selectivity of heme-catalyzed GAPDH tyrosine nitration. The modulation is associated with the hydrophilicity of the Aβ peptides, which changed the surrounding environment of the heme. At the same time, the Aβ-heme complexes were found to be more effective at inactivating GAPDH than heme alone, and the selective tyrosine nitration that was catalyzed by Aβ-heme played an important role. These findings suggest an alternative mechanism for the selectivity of protein tyrosine nitration, which may lead to a better understanding of the factors that influence protein tyrosine nitration selectivity and the important roles of Aβ and heme in the pathogenesis of Alzheimer's disease, where Aβ accumulation and Aβ-dependent protein nitration play central roles.     

Effect of insulin nitration by peroxynitrite on its biological activity

Insulin may be a potential target of ONOO- during conditions involving accelerated rates of oxygen radical and nitric oxide generation in pancreatic islet beta-cells. To evaluate the effect of protein tyrosine nitration on insulin function, the porcine insulin was nitrated by ONOO-. Insulin nitration was confirmed by spectral changes, Native-PAGE, and mass spectrometry. The bioassay results show that mono-nitro-insulin affected its receptor binding and hypoglycemic capacities to a certain extent, but still hold high biological activities.     

Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.     

Site-specific nitration differentially influences tau 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.     

GAPDH enhances group II intron splicing in vitro

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.