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EB2003 SYMPOSIUM
Mitochondrial Nitric Oxide
Department of 1Chemistry, 2Biology, and 3Biochemistry and Molecular Biology, University of Minnesota, Duluth, Minnesota 55812
Submitted 11 August 2003 ; accepted in final form 28 August 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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nitrotyrosine; mitochondria; oxidative stress; aging; proteomics
-isoform of neuronal NOS, with a different pattern of acylation from the endothelial NOS and phosphorylated at the COOH-terminal end (13). Nitric oxide is produced by mitochondria at a rate of 2 nmol·min–1·mg protein–1; this molecule interacts with cytochrome oxidase depending on the [NO·]-to-[O2] ratio, modulating the respiratory rate (18, 19), extending the oxygen gradient in tissues (17). During this process, ATP production (18) and oxygen free radicals generation (45) are also modulated by the NO·-cytochrome oxidase interaction. The relevance of this production relies on the effect of NO· on cytochrome oxidase (9, 11, 18), a target different from the well-known effects mediated through the activation of soluble guanylate cyclase (37).
Besides this physiological effect of modulating the availability of cellular oxygen, an overproduction of NO· or a sustained NO· generation may lead to pathological processes. For example, it has been demonstrated that a sustained mitochondrial NO· generation is required to promote cytochrome c release and the eventual activation of apoptosis (16). This effect has been attributed to the effect of peroxynitrite on the pyridine nucleotide-linked calcium release (16, 48). The apparent contribution of peroxynitrite and other reactive nitrogen oxides (such as nitrogen dioxide, dinitrogen trioxide, and other nitrogen oxides of the general formula NOx) to various disease processes has prompted a renewed interest in the nitrogen and oxygen-related free radical chemistry (4, 26, 31). These species can each react with a variety of cellular targets in vitro. Among them, L-tyrosine could be converted partly to 3-nitro-L-tyrosine on exposure to various reactive nitrogen oxides (55), and formation of nitrotyrosine has been linked to various pathological states (22, 29, 30, 46, 50). Despite intensive investigation into altered activities of nitrated proteins, the mechanism of nitration in biological systems remains controversial. Two major pathways have been suggested to be responsible for tyrosine nitration in vivo, both requiring the exposure of the biological system to NO·, without indicating NO· as the direct nitrating species. These pathways involve either the nitrative chemistry of peroxynitrite (5) or the catalytic action of heme peroxidases using hydrogen peroxide and nitrite (8, 12).
It was the goal of this project to investigate whether the nitration of proteins in mitochondria were enhanced under conditions of a sustained NO· production. Nitration of proteins constitutes a posttranslational modification by which NO· may exhibit long-term, damaging effects above those bioregulatory ones mediated through the interaction with cytochrome oxidase. Our studies were aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such process in the cellular milieu. We investigated the localization of nitrated proteins, identified the nitrated proteins by proteomic approaches, modulated nitration by stimulating the mitochondrial production of NO·, and found a consensus sequence for nitration. These results allowed us to explore the mechanism of nitration and gain understanding of the role of nitrated proteins in mitochondrial function and ultimately in cell and organ function.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Mannitol, sucrose, EGTA, HEPES, fatty acid-free bovine serum albumin (BSA), cytochrome c, DEAE cellulose, ADP, ATP, NAD+, NADP+, NADPH, NADH, lactic dehydrogenase, aminotyrosine, nitrotyrosine, sodium dithionite, azocasein, and normal goat serum were from Sigma Chemical (St. Louis, MO). Goat anti-mouse IgG and mouse anti-nitrotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY). Goat anti-rabbit IgG was purchased from Transduction Laboratories (Lexington, KY). Hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were purchased from Worthington (Lakewood, NJ).
Biological Materials
Isolation and purification of mitochondria. Liver mitochondria were isolated from adult male Wistar rats using differential centrifugation followed by Percoll gradient purification as described before (20). The respiratory control ratio (RCR) and ADP-to-O ratio (P/O) number of the preparations used in this study were 5.0 (or above) and 1.5, respectively, determined with 10 mM succinate and 0.45 mM ADP in reaction medium (0.22 M sucrose, 50 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, and 10 mM HEPES/KOH, pH 7.4) using a Clark-type oxygen electrode (Hansatech Instruments; Norfolk, UK).
Subfractionation of mitochondria. The subfractionation of mitochondria was performed with a controlled digitonin incubation, followed by sonication of mitoplasts (40). To assess purity of the fractions, the activities of enzymatic markers were followed according to published procedures (10, 36, 42, 47) as indicated below.
Assay for Marker Enzymes of Submitochondrial Fractions
Assay for monoamine oxidase. Monoamine oxidase activity was measured spectrofluorometrically (315 and 410 nm, excitation and emission wavelengths, respectively) by coupling the production of H2O2 with the oxidation of p-hydroxyphenylacetic acid catalyzed by horseradish peroxidase. The reaction mixture contained 40 µM p-hydroxyphenylacetic acid, 5 U/ml horseradish peroxidase, 10 mM benzylamine, and 5–150 µg/ml mitochondrial protein in 70 mM potassium phosphate buffer, pH 7.2.
Assay for adenylate kinase activity. Adenylate kinase activity was determined spectrophotometrically at 340 nm by coupling the production of ADP to the oxidation of NADH via the reactions catalyzed by pyruvate kinase and lactic dehydrogenase. The reaction mixture contained 0.4 mM NADPH, 0.1 mM ATP, 0.1 mM phosphoenolpyruvate, 1 µM rotenone, 0.04 U/ml of each pyruvate kinase and lactate dehydrogenase, 0.1 mM AMP, 0.04% Lubrol, 6 mM MgCl2, 130 mM KCl, 0.1 M Tris·HCl (pH 7.5), and 20–400 µg/ml of mitochondrial protein.
Assay for cytochrome oxidase activity. Cytochrome oxidase activity was determined by spectrophotometrically measuring the oxidation of 50 µM ferrocytochrome c at 550 nm in a reaction mixture containing 0.25 M sucrose, 5 mM MgCl2, and 50 mM HEPES (pH 7.4), using 10–70 µg/ml of mitochondrial protein. Assay for malate dehydrogenase activity. This activity was determined by spectrophotometrically measuring the formation of NADH at 340 nm. The reaction mixture contained 0.5 mM NADH, 0.25 mM oxaloacetate, 4 µM rotenone, 80 µM Na2S, and 10–200 µg/ml mitochondrial protein in 25 mM potassium phosphate (pH 7.4).
Evaluation of Nitration Level in Mitochondria and Mitochondrial Fractions
Mitochondrial fractions were separated by SDS-PAGE under denaturing and reducing conditions by using 10% polyacrylamide gels. Samples (1.5–20 µg) and molecular weight markers (Amersham Pharmacia Biotech) were heated for 5 min at 95°C in sample buffer [10 mM Tris·HCl (pH 8.0), 1 mM EDTA, 2.5% (wt/vol) SDS, 2.5% mercaptoethanol, and 0.01% (wt/vol) bromphenol blue]. Gels were run with Laemmli buffer at 75 V for 20 min followed by 200 V for 1 h. Proteins were visualized with Coomassie blue or silver stain. Western blot analysis was performed by transferring proteins to a polyvinyliledine difluoride membrane 3 h to overnight at 40 V. Membranes to be probed for nitrotyrosine were blocked for 2 h in a solution containing 1% nonfat dry milk and 10% normal goat serum in Tris-buffered saline with Tween 20 [TBS-T; 150 mM NaCl, 10 mM Tris·HCl (pH 7.6), and 0.05% Tween 20]. These membranes were then incubated overnight with a mouse monoclonal antibody against nitrotyrosine (1/2,000 in blocking solution), washed in TBS-T, and then incubated for 30 min with goat antibodies against mouse IgG conjugated with horseradish peroxidase (1/20,000 in blocking solution). The immunocomplexes were developed using enhanced horseradish peroxidase-luminol chemiluminescence reaction detected with photographic film (Hyperfilm ECL; Amersham). Nitration was evaluated by scanning the films and processing the spots with the National Institutes of Health Image software. The films were obtained with gels loaded with a different amount of protein to ensure the linear response between chemiluminescence and protein. The specific nitration (defined in Table 2 as b/a) was evaluated by semiquantitative dot blots in a GIBCO-BRL Convertible Filtration Manifold System using nitrated BSA as a standard. Albeit not shown, comparable results were obtained using diaminobenzidine and hydrogen peroxide. Protein was determined by Lowry (35). Specific nitration (see Table 2) was evaluated by HPLC with fluorescence detection as explained below.
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Incubation Conditions
To evaluate protein nitration under conditions of endogenous NO· production, purified and coupled rat liver mitochondria were incubated with 1 mM L-Arg or NG-monomethyl-L-arginine (L-NMMA) in reaction medium supplemented with proteolytic inhibitors and a respiratory substrate. The proteolytic inhibitors were optimized to ensure that no significant proteolysis was occurring during the enhanced NO· production (unless half-life measurements of nitrated proteins were performed in which case no proteolytic inhibitors were added). To this end, 1 mg protein/ml mitochondria was incubated in the reaction medium supplemented with 0.5% fluorescein thiocyanate-labeled casein, L-Arg, or L-NMMA and with various concentrations of proteolytic inhibitors. The mixture was incubated at 23°C and protected from light exposure for 1 h. The reaction was stopped by adding 5% trichloroacetic acid, and the fluorescence of the supernatant was evaluated in a Shimadzu RF-1501 fluorometer (excitation wavelength 490 nm and emission wavelength 525 nm) following the procedure described by Twining (54). This assay indicated that the addition of 1.8 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1.5 mM aprotinine, 90 nM bestatin, 30 nM E-64 protease inhibitor, 40 nM leupeptin hemisulfate, 18 nM pepstatin A, and 0.2 mM of each EDTA and EGTA to 1 mg protein/ml stopped the proteolysis during the 1-h incubation time.
The reaction medium used to follow NO· production by purified mitochondria (1 mg protein) contained substrate (either malate-glutamate or succinate) and 0.1 mM oxymyoglobin. The oxidation of oxymyoglobin stimulated by L-Arg and sensitive to L-NMMA, an inhibitor of NOS, was followed at 581–592 nm at 22°C (20). After mitochondria were incubated at 25°C for the indicated time, if half-life measurements were required, mitochondria were pelleted and washed extensively to remove remaining L-Arg or L-NMMA. These preparations were further incubated in buffer plus 10 mM of a respiratory substrate for an additional 60, 120, 180, and 240 min. At each time point, mitochondrial proteins were analyzed by two-dimensional (2-D) gels (13). These gels were stained with SYPRO Ruby (Molecular Probes), and parallel ones were blotted and probed for nitrotyrosine. From 2-D gels, the spots corresponding to a 
-subunit of F0F1-ATPase, carbamoylphosphate synthetase I, and glutamate dehydrogenase were identified by isoelectric point (pI), molecular weight, and matrix-assisted laser desorption ionization with time of flight (MALDI-ToF) of trypsin fragments. To this end, proteomic maps of nitrated proteins in the mitochondria and in each of the subfractions were obtained by excising the protein spots that cross-reacted with anti-nitrotyrosine antibodies followed by in-gel digestion with trypsin and analyzing the fragments by MALDI-ToF at the Mass Spectrometry Consortium for the Life Science (Biochemistry Department, University of Minnesota). The proteomic maps were built using internal markers (loaded with the samples or using glutamate dehydrogenase, carbamoyl phosphate, and -subunit) to adjust for warping, and molecular weight and pI were evaluated using the software Phoretix 2-D Nonlinear Dynamics.
2-D Gel Electrophoresis
Mitochondrial fractions were separated by pI on precast gel strips with a linear gradient of pH 3–10 from Amersham Pharmacia Biotech (Piscataway, NJ) by using the Multiphor II with the Immobiline Strip tray. The immobilized pH gradient (IPG) strips were rehydrated overnight in rehydration buffer [6 M urea, 2 M thiourea, 2% Nonidet P-40, 2% IPG buffer (pH 3–10), and 0.1 M dithiotreitol]. Twenty grams of mitochondrial sample were solubilized in the rehydration buffer for 30 min at room temperature before the rehydration of the strips. The voltage was provided by a Hoefer power supply PS 3501 XL, and the program utilized was the following: 300 V for 1 min, followed by a linear gradient from 300 to 3,500 V for 1.5 h and fixed at 3,500 V for 4.5 h. The strips were preequilibrated with 6 M urea, 30% glycerol, 1% SDS, 5 mg/ml dithiothreitol, and 50 mM Tris (pH 6.8) for 10 min at room temperature. They were then loaded onto 10% SDS-PAGE and run in a Hoefer SE600 overnight at 50 V. Gels were electroblotted and analyzed with antibodies to nitrotyrosine by Western blot as described above. In some instances, the blots probed for nitrotyrosine were stripped, washed three times for 10 min in TBS-T, washed twice for 5 min in 100 ml 0.4% Tween 20 in PBS, and stained with 0.1 ml India ink in 100 ml 0.3% Tween in PBS. The blots were incubated for 4–6 h and destained with PBS. This procedure differs from that used by Aulak et al. (1) in the following conditions. First, a total transfer of proteins from 2-D gels to membranes was sought, as opposed to a partial transfer (1). This latter procedure may favor the selective transfer of certain proteins over others (e.g., most abundant), thus creating a nonrepresentative sampling of all nitrated proteins. Furthermore, total transfer ensured maximum recovery favoring high signal-to-noise ratios when subsequent analyses were performed. Second, the efficiency of the transfer was checked by staining the gels, after the blotting was performed, with SYPRO Ruby to evaluate the presence of remaining proteins. Third, the transfers were performed with two parallel membranes to ensure that some proteins were not electrodiffusing beyond the first membrane. Finally, to find the protein sequences containing the nitro moieties, mass spectrometry analyses were performed on proteins from mitochondrial subfractions (as opposed to those obtained from whole mitochondria) to increase their concentration, improving the chances of obtaining enough material to detect the nitrated sequence.
High-Performance Liquid Chromatography with Fluorescence Detection for Evaluation of Nitrotyrosine in Mitochondria
Samples of mitochondria were digested with proteinase K [1:20 (wt/wt), enzyme:protein] in 0.2 M phosphate buffer (pH 7.4) for 2–3 h at 50°C, treated with a second equal aliquot, and allowed to incubate for an additional 12–16 h. To ensure an efficient proteolysis, these conditions were optimized by following the proteinase K-catalyzed proteolysis of azocasein. Proteolytic activity was evaluated by using 1 mg/ml azocasein incubated with 1 mg/ml mitochondrial protein digested with proteinase K as described above. On completion of proteolysis, 10% trichloroacetic acid was added to the hydrolysate, followed by centrifugation for 10 min at 12,000 g. The supernatants were analyzed by following the absorbance at 390 nm. The observed absorbance measurements were compared with those of azocasein digested alone (100% digestion) and azocasein without proteinase K (0% proteolysis) (49).
After digestion of mitochondrial samples, they were divided into thirds. One was left untreated, the second one was treated with 60 mM sodium dithionite to reduce nitrotyrosine to aminotyrosine, and the third was reduced with the same concentration of sodium dithionite and spiked with a known amount (10 pmol) of aminotyrosine. The digested samples were analyzed by reversed-phase HPLC using two C18 columns (4.6 x 250 mm) inline. Mobile phase A consisted of 50 mM sodium citrate and 50 mM acetic acid (pH 3.1), and mobile phase B was composed of 10% methanol, 50 mM sodium citrate, and 50 mM acetic acid (pH 3.1). For the elution of the compounds, the following linear gradient program was followed at a flow rate of 0.8 ml/min: from 100 to 98% phase A in 10 min, from 98 to 50% phase A in 20 min, from 50 to 25% phase A in 25 min, and then from 25 to 0% phase A in 40 min. The eluted fractions were monitored with a diode array and fluorescence detectors (excitation wavelength = 277 nm and emission wavelength = 307 nm). The quantification was performed by taking the area of the peak identified as aminotyrosine and interpolating the values in a plot performed with various concentrations of purified aminotyrosine. Given that the limit of detection of this method was below 3 pmol/mg mitochondrial protein, the protein concentration of the original samples was adjusted to fall above this limit within the linear response of the method.
Statistical Analysis
The results are means ± SD of three replicates from one experiment. Each experiment was carried out a minimum of three times, and the results shown represent all results obtained. The effect of treatment was compared with control values by one-way analysis of variance. Tests were carried out using StatSimple version 2.0.5 from Nidus Technologies (Toronto, Ontario, Canada).
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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To investigate the submitochondrial distribution of nitrated proteins in the rat liver mitochondria, Percoll-purified organelles were separated into fractions by using a controlled digitonin treatment followed by sonication of mitoplasts (See MATERIALS AND METHODS). The purity of each fraction; i.e., outer membrane (OM), intermembrane space (IMS), inner membrane (IM), contact sites (CS), and matrix (M), was assessed by measuring the specific activities of various enzymatic markers (Table 1). The characterization of these fractions indicated that they were suitable for our studies because cross contamination was minimal and each of them was significantly enriched by their corresponding marker (Table 1).
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Equal protein amounts of each submitochondrial fraction were separated by SDS-PAGE (Fig. 1) and stained with Coomassie blue (Fig. 1A). Western blots performed with antibodies to nitrotyrosine resulted in the appearance of numerous bands in all fractions (Fig. 1B). No bands were obtained if the blots were previously blocked with 10 mM nitrotyrosine, if the primary antibody were omitted from the incubation, or if the blots were previously treated with 10 mM dithionite in bicarbonate buffer (pH 9.0). The transfer efficiency of the proteins from the gel to the membrane was equal to all the proteins (under our conditions) because it was evaluated by staining the gel, after blotting, with Coomassie blue. These experiments demonstrated unequivocally that rat liver mitochondria (and all the subfractions derived from them) were endowed with nitrated proteins.
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Of note, neither all the proteins nor the most abundant ones present in each fraction were nitrated (compare Fig. 1, A and B). The fraction that exhibited the highest nitration was M, followed by IM, IMS, CS, and OM (Table 2). This pattern of nitration did not follow a linear correlation with protein distribution (Table 2). The specific nitration (expressed as nmol nitrotyrosine/g protein), evaluated by HPLC, was higher in the particulate (CS, OM, and IM) than in soluble (M and IMS) fractions (Table 2, 11 ± 2 and 2.5 ± 0.3 nmol/g, respectively; P < 0.01), although 86% of the total protein was present in the soluble fractions.
Protein Half-Lives and Nitration
It could be surmised that the fractions containing longer-lived proteins exhibited the higher level of nitration, given that proteins always exhibit a "background" of oxidative stress-related modifications. However, the specific nitration did not correlate with the mean half-lives of mitochondrial proteins (Table 2), indicating that the longer-lived proteins do not necessarily have higher chances of getting nitrated.
Despite the lack of correlation between nitration and average half-life, it seemed appropriate to investigate individual proteins given the vast heterogeneity in the turnover rate of mitochondrial proteins (from 24 to >100 h; see Refs. 27 and 28). To this end, pulse experiments were designed to determine the half-lives of three representative nitrated proteins.
We studied the effect of nitration on the turnover of carbamoyl phosphate synthetase I, -subunit of F0F1-ATPase, and glutamate dehydrogenase. These proteins became significantly nitrated evaluated by Western blotting over time under conditions of a sustained, endogenous mitochondrial NO· production (Fig. 2), have different half-lives, are relative abundant (they represent 18, 3, and 5%, respectively, of the mitochondrial protein), and are encoded by the nuclear genome, excluding the possibility of de novo protein synthesis throughout our experiments.
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The results depicted in Fig. 2 demonstrate that when mitochondria are producing NO·, nitration of proteins becomes evident after 15–30 min of incubation, indicating the need for a sustained production. It could be noted that the nitration rate seemed to be characteristic of each protein, not relying on molecular weight, abundance, pI, or half-life (Fig. 2 and Table 3).
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On evaluation of the half-lives of the nitrated proteins, our results indicated that the nitration increased the protein turnover significantly from essentially days to hours (Table 3), as also observed by others (51), suggesting that nitrative modifications (as it has been indicated for oxidatively modified proteins; Ref. 21) may trigger the recognition and/or activation of mitochondrial proteolytic enzymes. It must be questioned whether our experimental conditions (isolated mitochondria incubated with different substrates) had artificially enhanced the degradation of all mitochondrial proteins without indicating a preferential degradation of those nitrated. However, the half-lives of native proteins obtained in this study were not significantly different from those obtained under in vivo conditions (Table 3), excluding a nonspecific activation of proteolysis under our experimental conditions.
The increased turnover of nitrated proteins challenges the putative role of this posttranslational modification in signal transduction pathways (23, 58), because classic phosphorylation/dephosphorylation processes are reversible and do not require proteolysis of the entire protein to activate/deactivate pathways.
Nitrated Proteins and Metabolic Pathways
From our previous results, it seemed that nitration of proteins was favored for proteins located close to or at the mitochondrial membranes. Thus we investigated whether nitrated proteins belonged to a specific metabolic pathway(s). If the nitrating agent would have been produced at the vicinity of a certain protein, thus other constituent proteins of that pathway were expected to become nitrated given that enzymes of certain pathways are physically associated with one another to facilitate substrate channeling between active sites and, therefore, enhance its efficiency (metabolon hypothesis; see Refs. 39, 41, and 52).
To test this hypothesis, mitochondrial proteins were separated by 2-D gel electrophoresis, blotted, and immunoprobed for nitrotyrosine. The positive spots were trypsinized and analyzed by MALDI-ToF. With the use of peptide mass fingerprinting data, molecular weight, pI, organelle, and species, 37 proteins were positively identified (Table 4), whereas another four proteins presented no known matches in the Swiss-Prot or TrEMBL data bases. Those proteins (12 proteins) of which the nitration increased significantly (twofold minimum) during a sustained, endogenous NO· production (L-Arg-supplemented mitochondria) are indicated by asterisks in Table 4. Of note, a small fraction of the proteins found in this study were identified as nitrated by another study (1). The reasons for this apparent discrepancy are the intrinsic characteristics of each biological model and experimental conditions for analyzing nitrated proteins (see MATERIALS AND METHODS).
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Generally, our results indicated that, although most (78.4%) nitrated proteins were part of the metabolic pathways [as opposed to (16.2%) energy transduction and (5.4%) structural functions], there was not a particular pathway in which all its mitochondrial constituents were nitrated. Moreover, the distribution of proteins was the same (27.6%) between lipid and carbohydrate metabolism, followed by (13.8%) amino acid, (10.3%) ammonia, (10.3%) heme, (6.9%) ethanol, and (3.5%) hydroperoxide metabolic pathways. Some pathways in which a close proximity of the constituents was expected and reported (55), e.g., the tricarboxylic acid cycle, infrequent nitration was observed among its constituents (e.g., citrate synthase, isocitrate dehydrogenase, and fumarase were nitrated from 8 tricarboxylic acid cycle enzymes). However, we found three exceptions to this general observation: the urea cycle (in which both mitochondrial components were nitrated, i.e., carbamoyl phosphate synthetase I and ornithine carbamoylase and three enzymes of 5 enzymes outside of the cycle but required for its operativity, i.e., glutamate dehydrogenase, fumarase, and carbonic anhydrase), the heme biosynthetic pathway (in which 3 of 4 mitochondrial components were nitrated except for protoporphyrinogen oxidase), and the pyruvate dehydrogenase complex (in which E1, E2, and E3 were nitrated). These observations may indicate that the constituents of these pathways are forming a more packed hyperstructure (like the multienzyme complex of pyruvate dehydrogenase) than others, that the nitration process is more specific than expected, or a combination of both possibilities.
Considering that these latter pathways (i.e., urea cycle and associated enzymes, pyruvate dehydrogenase complex, and heme biosynthesis) were more affected by nitration than others, the potential exists to use the nitration of these pathways or their operativity (or if an association between nitration and loss of activity is found) as specific markers of nitrative stress.
Nitration and Target Sequence
Given that the nitration of proteins could not be ascribed to most parameters (i.e., molecular weight, pI, abundance, half-life, and metabolic pathway), studies were undertaken to see whether a specific sequence of amino acids was preferably nitrated to others under conditions of a sustained NO· production. To this end, several sequences of nitrated proteins (it was possible to get fragments containing the nitro moiety from 8 of the 12 proteins shown in Table 4) obtained by MALDI-ToF, and subsequently analyzed by tandem mass spectroscopy, were aligned using CLUSTALW to investigate the presence of a consensus sequence (Fig. 3). All 10 protein fragments showed the following pattern that includes the target tyrosine: [LMVI]-X-[DE]-[LMVI]-X(2,3)-[FVLI]-X(3,5)-Y, where X is any amino acid and Y is the target tyrosine. Considering that 5 of 11 fragments presented an additional [LMVA] residue after [FVLI] and that essentially hydrophobic residues were present at several positions along the pattern, a more general pattern was constructed of the form H-X-[DE]-H-X(2,3)-H(2)-X(2,4)-Y, in which H represents a hydrophobic residue (such as L, M, V, I, P, A, F, or W). Several reports (24, 56, 57, 59) provided evidence for selective nitration of Tyr at protein transmembrane or intramembrane domains. These previous reports support our findings that Tyr at hydrophobic pockets are preferred targets for nitration. Furthermore, it has been suggested that Tyr nitration is enhanced by close proximity of acidic residues and decreased when located adjacent to easily oxidizable amino acids such as Cys or Met (30, 34). In our case, the first observation was verified (the presence of D or E), but no experimental evidence was found to support the latter.
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When the consensus sequence was utilized to search the protein data base (within the rattus taxon, 130,251 entries), all eight nitrated proteins (those utilized to construct the pattern) were retrieved besides another six from Table 4, indicating that the sequence could be used as a predictor of nitration. To ascertain the specificity of the pattern, when the sequence was utilized to search in a randomized data base (ScanProsite software available at Expasy web site, "shuffle20" condition), four proteins from Table 4 were retrieved (only one from the original set used to deduce the sequence). Thus these results indicated the potential use of this pattern to predict target proteins for nitration. Of interest, it has been proposed that Tyr nitration may interfere with the phosphorylation/dephosphorylation of Tyr involved in signal transduction pathways. However, the consensus sequence for Tyr phosphorylation {[RK]-X(2)-[DE]-X(3)-Y or [RK]-X(3)-[DE]-X(2)-Y} is not similar to that required for nitration, challenging the concept that Tyr nitration may not halt Tyr phosphorylation under in vivo conditions, because different Tyr are expected to be targeted by these processes. This observation should not be extended to in vitro systems, in which the nitration of proteins utilizing various agents and under various experimental conditions (chemical characteristics of the nitrating agent, ratio of nitrating agent to protein, pH, among others) may substantially differ from the in vivo conditions, overcoming specificity issues.
Biological Impact of Protein Nitration
It must be questioned whether nitration of mitochondrial proteins modulated by a sustained, endogenous NO· production has any biological impact given that the turnover of these proteins was significantly accelerated on modification, implicating that the putative change(s) in activity ensuing nitration could be overcome by faster turnovers. However, experiments performed with 1.5-, 3-, and 10-mo-old animals indicated that the nitration of mitochondrial proteins accumulated with age (not shown). Particularly, the nitration of the -subunit of F0F1-ATPase, glutamate dehydrogenase, and carbamoyl phosphate synthetase I was 2, 3, and 22 times higher than that found in young rats (Table 5).
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If the association between chemically induced nitration and altered activity (38, 43) can be extended to these particular proteins, then it is expected that these pathways might be altered as the animals age (by a mechanism that may include an increased rate of NO· production, decreased rate of degradation of nitrated proteins, or a combination of both, among others) having a significant effect on energy metabolism and nitrogen disposal. Thus the faster turnover of nitrated proteins may limit the biological impact when short-term effects are sought; however, in certain pathophysiological situations (e.g., aging, altered proteolytic mechanisms) in which the normal balance between nitrated and native proteins is shifted, then effects on mitochondrial and cellular metabolism should be foreseen.
In conclusion, our study indicated that the nitration process may occur as a result of two conditions: 1) a primary sequence that allows the nitration of Tyr at the ortho position of the hydroxyl group, and 2) the localization of the protein at or close to the membrane. This latter condition may suggest that either the nitrating agent is formed at or close to the target sequence, or despite where the nitrating agent was originated, its hydrophobic nature facilitates the partition to the membrane (or to protein hydrophobic pockets) thus increasing the chances of nitrating target-containing proteins. The fact that a particular sequence was more nitrated than others may indicate the involvement of more specific reactions through less reactive radicals, such as nitrogen dioxide.
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ACKNOWLEDGMENTS |
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GRANTS
This research was supported by National Institutes of Health Grants ES-011407 and GM-66768 and partially by Cottrell Research Grant CC-5675 and Petroleum Research Fund (American Chemical Society Grant 37470-B4). Processing of two-dimensional gel images was performed at the Visualization and Imaging Laboratory, University of Minnesota.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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