Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.     

Investigation of local primary structure effects on peroxynitrite-mediated tyrosine nitration using targeted mass spectrometry

Protein-tyrosine nitration (PTN) is a posttranslational modification resulting from cellular nitrosative stress that has been implicated in a wide variety of disease states. Determination of factors that influence selectivity of PTN remains a major challenge due to several issues including low biological levels of PTN, proximity of target sites on a single analyte, and analytical limitations for site-specific quantification of the nitration modification. We report a systematic approach that addresses relevant contributing factors to PTN with particular focus on determining the effect of changing proximal amino acid side chain structure on tyrosine nitration yield. A trend was observed in which nitration yield tends to be greater when the tyrosine residue is surrounded by basic and/or uncharged polar residues compared to nitration levels observed when hydrophobic and acidic residues are proximal to the tyrosine residue. Moreover, an electric dipole effect was observed where a higher degree of charge asymmetry surrounding the tyrosine residue correlates with an increased tyrosine nitration yield in certain cases. The reported data are expected to facilitate site-specific prediction and validation of PTN, especially in cases of potential target residues that share a similar solvent exposure environment and contain elements of known higher-order structure.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.     

GPS-CCD: a novel computational program for the prediction of calpain cleavage sites

As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/.     

3-Nitrotyrosine modification of SERCA2a in the aging heart: a distinct signature of the cellular redox environment

In the aging heart, decreased rates of calcium transport mediated by the SERCA2a isoform of the sarcoplasmic reticulum (SR) Ca-ATPase are responsible for the slower sequestration of cytosolic calcium and consequent prolonged muscle relaxation times. We report a 60% decrease in Ca-ATPase activity in the senescent Fischer 344 rat heart relative to that of young adult hearts; this functional decrease can be attributed, in part, to the 18% lower abundance of SERCA2a protein. Here, we show that the additional loss of activity is a result of increased 3-nitrotyrosine modification of the Ca-ATPase. Age-dependent increases in nitration of cardiac SERCA2a are identified using multiple analytical methods. In the young (adult) heart 1 molar equivalent of nitrotyrosine is distributed over at least five tyrosines within the Ca-ATPase, identified as Tyr(122), Tyr(130), Tyr(497), Tyr(586), and Tyr(990). In the senescent heart, the stoichiometry of nitration increases by more than two nitrotyrosines per Ca-ATPase, coinciding with the appearance of nitrated Tyr(294), Tyr(295), and Tyr(753). The abundant recovery of native analogues for each of the nitrated peptides indicates partial modification of multiple tyrosines within cardiac SERCA2a. In contrast, within skeletal muscle SERCA2a, a homogeneous pattern of nitration appears, with full site (1 mol/mol) nitration of Tyr(753), in young, with additional nitration of Tyr(294) and Tyr(295), in senescent muscle. The nitration of these latter vicinal sites correlates with diminished transport function in both striated muscle types, suggesting that these sites provide a mechanism for downregulation of ATP utilization by the Ca-ATPase under conditions of nitrative stress.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Protein and lipid nitration: role in redox signaling and injury

Protein and lipid nitration represent novel footprints of oxidative and nitrative stress processes. In this review, we first discuss the mechanisms of formation of protein 3-nitrotyrosine and nitrated fatty acids as well as their key biological and signaling actions. Elevation of protein 3-nitrotyrosine levels is associated to tissue injury, and some specific nitrated proteins play a causative role in disease progression; on the other hand, the substantiation on the role of tyrosine nitration on redox signaling is rather scarce. Herein, we also provide evidence to support that the nitration of lipids (i.e. to nitrofatty acids) results in the formation of novel endogenous modulators of redox processes, partially counteracting pro-inflammatory effects of oxidant exposure.     

Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dependent cholesterol efflux from macrophages

We recently reported that apolipoprotein A-I (apoA-I), the major protein component of high density lipoprotein, is a selective target for myeloperoxidase (MPO)-catalyzed nitration and chlorination in both and serum of subjects with cardiovascular disease. We further showed that the extent of both apoA-I nitration and chlorination correlated with functional impairment in reverse cholesterol transport activity of the isolated lipoprotein. Herein we used tandem mass spectrometry to map the sites of MPO-mediated apoA-I nitration and chlorination in vitro and in vivo and to relate the degree of site-specific modifications to loss of apoA-I lipid binding and cholesterol efflux functions. Of the seven tyrosine residues in apoA-I, Tyr-192, Tyr-166, Tyr-236, and Tyr-29 were nitrated and chlorinated in MPO-mediated reactions. Site-specific liquid chromatography-mass spectrometry quantitative analyses demonstrated that the favored modification site following exposure to MPO-generated oxidants is Tyr-192. MPO-dependent nitration and chlorination both proceed with Tyr-166 as a secondary site and with Tyr-236 and Tyr-29 modified only minimally. Parallel functional studies demonstrated dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid binding activities with apoA-I modification by MPO. Finally tandem mass spectrometry analyses showed that apoA-I in human atherosclerotic tissue is nitrated at the MPO-preferred sites, Tyr-192 and Tyr-166. The present studies suggest that site-specific modifications of apoA-I by MPO are associated with impaired lipid binding and ABCA1-dependent cholesterol acceptor functions, providing a molecular mechanism that likely contributes to the clinical link between MPO levels and cardiovascular disease risk.     

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.     

Nitration and oligomerization of tau induced by peroxynitrite inhibit its microtubule-binding activity

Abnormally nitrated tau has been found recently in the neurofibrillary tangles of Alzheimer's disease (AD). However, whether and how nitration of tau is involved in AD pathology is not known. Herein, we found that in vitro incubation of peroxynitrite with recombinant tau resulted in nitration and oligomerization of tau in a dosage-dependent manner. Moreover, the nitrated tau showed a significantly decreased binding activity to taxol-stabilized microtubulesin in vitro. Further study demonstrated that peroxynitrite also induced tau nitration in neuroblastoma N2a cell line, and the nitrated tau was accumulated in the cells. We conclude that abnormal nitration of tau contributes to the impaired biological activity of tau in binding to the microtubules and the aggregation of tau, implying a novel mechanism responsible for the neurodegeneration seen in AD brain.     

Aspects, mechanism, and biological relevance of mitochondrial protein nitration sustained by mitochondrial nitric oxide synthase

The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO.-) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO.- exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO.- production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO.- (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration.     

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.     

Post-translational modifications of superoxide dismutase

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications.     

Protein tyrosine nitration--functional alteration or just a biomarker?

Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.     

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.     

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l ‐tryptophan. To develop cost‐efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self‐sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4‐F‐dl ‐tryptophan and 5‐F‐l ‐tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE‐based biocatalysts for the production of valuable nitroaromatics.     

In vivo protein tyrosine nitration in Arabidopsis thaliana

Nitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, based on an anti-3-nitroY antibody, was performed to pull-down potential in vivo targets of Y nitration in the Arabidopsis thaliana proteome. Further shotgun liquid chromatography-mass spectrometry (LC-MS/MS) proteomic analysis of the immunoprecipitated proteins allowed the identification of 127 proteins. Around 35% of them corresponded to homologues of proteins that have been previously reported to be Y nitrated in other non-plant organisms. Some of the putative in vivo Y-nitrated proteins were further confirmed by western blot with specific antibodies. Furthermore, MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of protein spots, separated by two-dimensional electrophoresis from immunoprecipitated proteins, led to the identification of seven nitrated peptides corresponding to six different proteins. However, in vivo nitration sites among putative targets could not be identified by MS/MS. Nevertheless, an MS/MS spectrum with 3-aminoY318 instead of the expected 3-nitroY was found for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Reduction of nitroY to aminoY during MS-based proteomic analysis together with the in vivo low abundance of these modifications made the identification of nitration sites difficult. In turn, in vitro nitration of methionine synthase, which was also found in the shotgun proteomic screening, allowed unequivocal identification of a nitration site at Y287.     

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.