Site-specific Nitration Research Articles

Manganese- and Platinum-Driven Oxidative and Nitrosative Stress in Oxaliplatin-Associated CIPN with Special Reference to Ca4Mn(DPDP)5, MnDPDP and DPDP.

Platinum-containing chemotherapeutic drugs are efficacious in many forms of cancer but are dose-restricted by serious side effects, of which peripheral neuropathy induced by oxidative-nitrosative-stress-mediated chain reactions is most disturbing. Recently, hope has been raised regarding the catalytic antioxidants mangafodipir (MnDPDP) and calmangafodipir [Ca4Mn(DPDP)5; PledOx®], which by mimicking mitochondrial manganese superoxide dismutase (MnSOD) may be expected to overcome oxaliplatin-associated chemotherapy-induced peripheral neuropathy (CIPN). Unfortunately, two recent phase III studies (POLAR A and M trials) applying Ca4Mn(DPDP)5 in colorectal cancer (CRC) patients receiving multiple cycles of FOLFOX6 (5-FU + oxaliplatin) failed to demonstrate efficacy. Instead of an anticipated 50% reduction in the incidence of CIPN in patients co-treated with Ca4Mn(DPDP)5, a statistically significant increase of about 50% was seen. The current article deals with confusing differences between early and positive findings with MnDPDP in comparison to the recent findings with Ca4Mn(DPDP)5. The POLAR failure may also reveal important mechanisms behind oxaliplatin-associated CIPN itself. Thus, exacerbated neurotoxicity in patients receiving Ca4Mn(DPDP)5 may be explained by redox interactions between Pt2+ and Mn2+ and subtle oxidative-nitrosative chain reactions. In peripheral sensory nerves, Pt2+ presumably leads to oxidation of the Mn2+ from Ca4Mn(DPDP)5 as well as from Mn2+ in MnSOD and other endogenous sources. Thereafter, Mn3+ may be oxidized by peroxynitrite (ONOO-) into Mn4+, which drives site-specific nitration of tyrosine (Tyr) 34 in the MnSOD enzyme. Conformational changes of MnSOD then lead to the closure of the superoxide (O2•-) access channel. A similar metal-driven nitration of Tyr74 in cytochrome c will cause an irreversible disruption of electron transport. Altogether, these events may uncover important steps in the mechanism behind Pt2+-associated CIPN. There is little doubt that the efficacy of MnDPDP and its therapeutic improved counterpart Ca4Mn(DPDP)5 mainly depends on their MnSOD-mimetic activity when it comes to their potential use as rescue medicines during, e.g., acute myocardial infarction. However, pharmacokinetic considerations suggest that the efficacy of MnDPDP on Pt2+-associated neurotoxicity depends on another action of this drug. Electron paramagnetic resonance (EPR) studies have demonstrated that Pt2+ outcompetes Mn2+ and endogenous Zn2+ in binding to fodipir (DPDP), hence suggesting that the previously reported protective efficacy of MnDPDP against CIPN is a result of chelation and elimination of Pt2+ by DPDP, which in turn suggests that Mn2+ is unnecessary for efficacy when it comes to oxaliplatin-associated CIPN.

Site-specific tyrosine nitration of group 1 allergens of house dust mite Dermatophagoides farinae (Der f 1) and Dermatophagoides pteronyssinus (Der p 1) in indoor dusts

Nitration can enhance the allergenicity of proteins. The nitration status of house dust mite (HDM) allergens in indoor dusts, however, remains to be elucidated. In the study, site-specific tyrosine nitration degrees of the two important HDM allergens Der f 1 and Der p 1 in indoor dust samples were investigated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The measured concentrations of native and nitrated allergens in the dusts were in the range of 0.86–29 μg g−1 for Der f 1 and from below the detection limit to 29 μg g−1 for Der p 1. Site-specific analysis revealed that all ten tyrosine residues in Der f 1 and Der p 1 were nitrated to different degrees in the investigated samples. The preferred nitration sites were Y56 in Der f 1 and Y37 in Der p 1 with the nitration degrees of 7.6–84% and 17–96% among the detected tyrosine residues, respectively. The measurements reveal high site-specific nitration degrees for tyrosine in Der f 1 and Der p 1 detected in the indoor dust samples. Further investigations are required to find out if the nitration really aggravates the health effects of HDM allergens and if the effects are tyrosine site-dependent.

A new route for the synthesis of 1-deazaguanine and 1-deazahypoxanthine.

Imidazopyridines and pyrrolopyrimidines are an important class of compounds in medicinal chemistry. They can also be considered as deaza-modified purine nucleobases, and as such have attracted a lot of interest recently in the context of RNA atomic mutagenesis. In particular, for 1-deazaguanine (c1G base), a significant increase in demand is apparent. Synthetic access is challenging and the few reports found in the literature suffer from the requirement of hazardous intermediates and harsh reaction conditions. Here, we report a new six-step synthesis for c1G base, starting from 6-iodo-1-deazapurine. The key transformations are copper catalyzed C-O-bond formation followed by site-specific nitration. A further strength of our route is divergency, additionally enabling the synthesis of 1-deazahypoxanthine (c1I base).

Site-Specific Nitration of Hsp90 Alters Cell Metabolism and Supports Schwannoma Cell Proliferation

Site-Specific Nitration of Hsp90 Alters Cell Metabolism and Supports Schwannoma Cell Proliferation

Structural Changes Induced in Hsp90 by Nitration lead to a Pathological Gain‐of‐function

Heat Shock Protein (Hsp90) is a pro‐survival molecular chaperone essential for the proper folding of a wide range of cellular proteins. It is highly abundant in most cell types and exists as a dimer that interacts with the client proteins. It is well described that the activity of Hsp90 is regulated by post‐translational modifications. We showed that in pathological conditions in which the powerful oxidant peroxynitrite is produced, Hsp90 undergoes nitration at 5 out of 24 tyrosine residues (Y) in its sequence. Further, nitration at Y33 and/or Y56 induces a pathological gain‐of‐function in Hsp90, leading to decreased mitochondrial activity and activation of the purinergic P2X7 receptor, which in motor neurons induces cell death. This is the first nitrated protein with a pathological function described to date. Nitration of Hsp90 is of particular relevance in amyotrophic lateral sclerosis (ALS), a motor neuron degenerative condition with an elusive pathogenesis and scant treatment options. We discovered that Hsp90 is endogenously nitrated in motor neurons in the spinal cord of ALS patients, the cell type that is compromised in this disease process. Establishing the structural changes induced in Hsp90 by nitration that lead to the pathological toxic function is crucial for the development of novel therapeutic strategies for ALS treatment. Here, using a combination of biophysical approaches we show that nitration of Hsp90 at Y33 and Y56, the residues relevant to the toxic function, had a profound impact on Hsp90 structure. To dissect the contribution of nitration at Y33 and/or Y56 on Hsp90 structural changes, we performed analytical ultracentrifugation sedimentation velocity experiments using recombinant Hsp90, peroxynitrite‐treated Hsp90 (fully oxidized protein), and site‐specific nitrated Hsp90 produced by genetic code expansion, carrying nitrotyrosine at either position 33 or 56, or simultaneously at both positions as the sole modification in the protein. Following peroxynitrite treatment, Hsp90 dimer was destabilized, showing a significant increase in the number of monomers, and the formation of oligomeric species. These results were confirmed by negative stain cryo‐electron microscopy, and size exclusion chromatography followed by multiangle light scattering. Site‐specific nitration at Y33 or Y56 was enough to destabilize the dimer conformation, while simultaneous nitration at Y33 and Y56 most closely resembled the structural changes observed after peroxynitrite treatment. In contrast, replacement of Y33 and Y56 by nitration‐resistant phenylalanine decreased dimer destabilization and prevented oligomer formation after peroxynitrite treatment. Together, these results suggest that nitration at Y33 and Y56 is enough to significantly affect the global structure of Hsp90, and that these changes may be responsible for nitrated Hsp90's toxic gain‐of‐function. Thus, targeting these structural changes may lead to the development of drugs that selectively inhibit nitrated Hsp90's pathological activity in ALS, without affecting the function of the unmodified chaperone in normal tissues.

Protein Tyrosine Nitration in Plant Nitric Oxide Signaling.

Nitric oxide (NO), which is ubiquitously present in living organisms, regulates many developmental and stress-activated processes in plants. Regulatory effects exerted by NO lies mostly in its chemical reactivity as a free radical. Proteins are main targets of NO action as several amino acids can undergo NO-related post-translational modifications (PTMs) that include mainly S-nitrosylation of cysteine, and nitration of tyrosine and tryptophan. This review is focused on the role of protein tyrosine nitration on NO signaling, making emphasis on the production of NO and peroxynitrite, which is the main physiological nitrating agent; the main metabolic and signaling pathways targeted by protein nitration; and the past, present, and future of methodological and strategic approaches to study this PTM. Available information on identification of nitrated plant proteins, the corresponding nitration sites, and the functional effects on the modified proteins will be summarized. However, due to the low proportion of in vivo nitrated peptides and their inherent instability, the identification of nitration sites by proteomic analyses is a difficult task. Artificial nitration procedures are likely not the best strategy for nitration site identification due to the lack of specificity. An alternative to get artificial site-specific nitration comes from the application of genetic code expansion technologies based on the use of orthogonal aminoacyl-tRNA synthetase/tRNA pairs engineered for specific noncanonical amino acids. This strategy permits the programmable site-specific installation of genetically encoded 3-nitrotyrosine sites in proteins expressed in Escherichia coli, thus allowing the study of the effects of specific site nitration on protein structure and function.

PredNTS: Improved and Robust Prediction of Nitrotyrosine Sites by Integrating Multiple Sequence Features.

Nitrotyrosine, which is generated by numerous reactive nitrogen species, is a type of protein post-translational modification. Identification of site-specific nitration modification on tyrosine is a prerequisite to understanding the molecular function of nitrated proteins. Thanks to the progress of machine learning, computational prediction can play a vital role before the biological experimentation. Herein, we developed a computational predictor PredNTS by integrating multiple sequence features including K-mer, composition of k-spaced amino acid pairs (CKSAAP), AAindex, and binary encoding schemes. The important features were selected by the recursive feature elimination approach using a random forest classifier. Finally, we linearly combined the successive random forest (RF) probability scores generated by the different, single encoding-employing RF models. The resultant PredNTS predictor achieved an area under a curve (AUC) of 0.910 using five-fold cross validation. It outperformed the existing predictors on a comprehensive and independent dataset. Furthermore, we investigated several machine learning algorithms to demonstrate the superiority of the employed RF algorithm. The PredNTS is a useful computational resource for the prediction of nitrotyrosine sites. The web-application with the curated datasets of the PredNTS is publicly available.

Design, synthesis and biological assessment of N-adamantyl, substituted adamantyl and noradamantyl phthalimidines for nitrite, TNF-α and angiogenesis inhibitory activities

A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.

Elucidating the Role of Site-Specific Nitration of α-Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified α-synuclein (α-syn). Despite advances in the chemical synthesis of α-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of α-syn. Here we report, for the first time, the site-specific incorporation of 3-nitrotyrosine at different regions of α-syn using native chemical ligation combined with a novel desulfurization strategy. This strategy enabled us to investigate the role of nitration at single or multiple tyrosine residues in regulating α-syn structure, membrane binding, oligomerization, and fibrils formation. We demonstrate that different site-specifically nitrated α-syn species exhibit distinct structural and aggregation properties and exhibit reduced affinity to negatively charged vesicle membranes. We provide evidence that intermolecular interactions between the N- and C-terminal regions of α-syn play critical roles in mediating nitration-induced α-syn oligomerization. For example, when Y39 is not available for nitration (Y39F and Y39/125F), the extent of cross-linking is limited mostly to dimer formation, whereas mutants in which Y39 along with one or multiple C-terminal tyrosines (Y125F, Y133F, Y136F and Y133/136F) can still undergo nitration readily to form higher-order oligomers. Our semisynthetic strategy for generating site-specifically nitrated proteins opens up new possibilities for investigating the role of nitration in regulating protein structure and function in health and disease.

Method to study stoichiometry of protein post-translational modification.

We report a new method to study stoichiometry of protein post-translational modification (PTM) via manipulating small-molecule labels. Such labels can simultaneously bind with all the modified amino acids in a target protein, resulting in a large signal. However, if the small-molecule labels are previously attached with a macromolecule comparable to the target protein in size, binding of one such label will prevent subsequent binding of more labels of its kind; thus, only one of the modified amino acids in the target protein can be bound with the label, resulting in a small signal. Therefore, the stoichiometric number of PTM can be determined by a ratio of the two signals. The proposed method can analyze nitration of several essential regulatory proteins of cellular life, all of which are found to have an integer stoichiometric number indicating regulated site-specific nitration. These results validate the efficacy of our method to provide detailed information on PTMs with biological significance.

Oral 1822-2 - Site-specific nitration of apolipoprotein A-I at tyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional

Oral 1822-2 - Site-specific nitration of apolipoprotein A-I at tyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼ 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Separation and characterization of nitrated variants of the major birch pollen allergen by CZE‐ESI‐μTOF MS

A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants.

High density lipoprotein is targeted for oxidation by myeloperoxidase in rheumatoid arthritis

ObjectivePhagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we...

Peroxynitrite-Mediated Oxidative Modifications of Complex II: Relevance in Myocardial Infarction

Increased O(2)(*-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO(-)) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved in OONO(-)-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO(-). This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis. Nitration of Y(56) and Y(142) was previously reported. Further analysis revealed that C(267), C(476), and C(537) are involved in OONO(-)-mediated S-sulfonation. To identify the disulfide formation mediated by OONO(-), nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C(306)-C(312), C(439)-C(444), and C(288)-C(575), were involved in OONO(-)-mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO(-)-dependent DMPO adduct was detected, and further LC-MS/MS analysis indicated C(288) and C(655) were involved in DMPO binding. These results offered a complete profile of OONO(-)-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.

Protein Tyrosine Nitration of the Flavin Subunit Is Associated with Oxidative Modification of Mitochondrial Complex II in the Post-ischemic Myocardium

Increased O(2)* and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart ( Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654 ). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide ((77)AAFGLSEAGFNTACVTK(93)) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.

Nitrite-Mediated Inactivation of Human Plasma Paraoxonase-1: Possible Beneficial Effect of Aromatic Amino Acids

Paraoxonase-1 (PON-1) is a high-density-lipoprotein-bound enzyme, and its major function is to prevent oxidation of low-density lipoprotein. Atherogenesis could be related to decreased activity of this enzyme. Nitrites (NO2-), either present as a contaminant and/or the main metabolic end product of nitric oxide (NO) degradation, may trigger nitrative damage to PON-1 enzyme. Minimal information is available concerning the effect of nitrite on the enzyme activity and the mechanism which it exerts its effect. The aim of this study was to analyze whether nitrites could play a role in modifying human PON-1 activity. Our results revealed that PON-1 activity was inhibited by nitrite in dose- and time-dependent manner. Site-specific nitration focused on phenolic residues, particularly tyrosine residues of the enzyme, may result in modification of its biological functions. Nitration of phenolic residues occurs via peroxynitrite (ONOO(-)) formation, which requires peroxides and nitrite. Thus, we tested the presence of peroxides, which are found in all plasma samples regardless of nitrite concentration. The inhibition of PON-1 activity by nitrite was significantly reduced by tryptophan, reduced glutathione (GSH), and catalase additions. Therefore, we concluded that nitrites may have a role in the inactivation of PON-1, probably through nitration of enzyme phenyl residues, and additions of individual aromatic amino acids, with highlighting on tryptophan, could be of important value in minimizing the nitrite-induced inhibition of PON-1 enzyme.

Nitration in Neurodegeneration: Deciphering the “Hows” “nYs”

Recent literature has ushered in a new awareness of the diverse post-translational events that can influence protein folding and function. Among these modifications, protein nitration is thought to play a critical role in the onset and progression of several neurodegenerative diseases. While previously considered a late-stage epiphenomenon, nitration of protein tyrosine residues appears to be an early event in the lesions of amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. The advent of highly specific biochemical and immunological detection methods reveals that nitration occurs in vivo with biological selectively and site specificity. In fact, nitration of only a single Tyr residue is often sufficient to induce profound changes in the activity of catalytic proteins and the three-dimensional conformation of structural proteins. Presumably, nitration modifies protein function by altering the hydrophobicity, hydrogen bonding, and electrostatic properties within the targeted protein. Most importantly, however, nitrative injury may represent a unifying mechanism that explains how genetic and environmental causes of neurological disease manifest a singular phenotype. In this review and synthesis, we first examine the pathways of protein nitration in biological systems and the factors that influence site-directed nitration. Subsequently, we turn our attention to the structural implications of site-specific nitration and how it affects the function of several neurodegeneration-related proteins. These proteins include Mn superoxide dismutase and neurofilament light subunit in amyotrophic lateral sclerosis, alpha-synuclein and tyrosine hydroxylase in Parkinson's disease, and tau in Alzheimer's disease.

Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method

In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2 V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85 V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO 2 for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23.