1Department of Medicine, University of Florida College of Medicine, and 2Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida
Submitted 7 July 2004 ; accepted in final form 17 November 2004
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ABSTRACT |
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S-nitrosylation; redox regulation
It is not clear how NO causes persistent inhibition of complex IV, but S-nitrosylation of key cysteine residues of complex IV may play a role. This notion is supported by the following observations. First, analysis of complex IV peptide sequences available in the Swiss-Prot database reveals that there are highly conserved cysteine residues across all species (sequences are available in the database) that are reported to be associated with the active center formation (30). Although it is still unclear whether NO can nitrosylate these cysteines to inhibit complex IV activity, NO has been reported to modulate the biological functions of many other intracellular signaling proteins by S-nitrosylation. These include two cysteine transcription factors, NF-B (22, 23) and activator protein-1 (AP-1) (62). Second, excessive NO may overwhelm the mitochondrial antioxidant systems, e.g., GSH and thioredoxin (Trx), leading to nitrosylation of critical thiols or preventing removal of NO from the already nitrosylated sulfhydryl (SH) moieties (16), which may also contribute to persistent inhibition of complex IV. This inhibition can be long-lasting (compared with NO-heme reactivity) and reversible (compared with tyrosine nitration-induced inhibition). Finally, it has been reported that NO inhibition of complex I and II of the mitochondrial electron transport chain in vivo results from S-nitrosylation of critical thiols and ONOO formation, respectively (9, 49). These observations suggest that critical/active site cysteine residues of complex IV may be unique targets for NO to nitrosylate and may serve as a novel molecular mechanism of NO-induced persistent inhibition of complex IV.
In the present study, pulmonary artery endothelial cells (PAEC) were used as a cell model to determine the effects of the slow-releasing NO donor 2,2'-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18) on persistent inhibition and S-nitrosylation of complex IV. Our results demonstrate that long-term exposure of cells to pathophysiological concentrations of NO causes persistent inhibition of complex IV, which is likely due to redox/S-nitrosylation of active site cysteine residues located in a putative NO-sensitive motif in complex IV.
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EXPERIMENTAL PROCEDURES |
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Cell culture and NO exposure. Cultured human (Clonetics, San Diego, CA) and porcine (primary cultures) PAEC were used as cell models to determine whether NO-induced persistent inhibition of complex IV is associated with oxidation/nitrosylation of complex IV S2. Porcine PAEC were obtained from the main pulmonary arteries of 6-mo-old pigs from a local slaughterhouse and were propagated in monolayer cultures as described by Zhang et al. (71, 73). Fifth to seventh passage cells in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics were used for all experiments.
In the present study, NOC-18 and PTIO were used as a NO donor and a NO scavenger, respectively. It has been reported that NO released from 1 mM NOC-18 results in steady-state levels of 15 µM NO in medium without any cofactors (Ref. 7, product technique data of Calbiochem). This is comparable to concentrations (130 µM) produced by endogenous inducible NOS in culture media and in plasma after cytokine stimulation or lung injury (47). NO concentrations used in the clinical arena for inhalation treatment of pulmonary hypertension, acute lung injury, and cardiopulmonary failure are 1530 ppm (510 µM) (4, 6, 8). Given that the NO concentrations to which the lung endothelium is exposed are slightly lower than that in the inhaled NO gas, exposure of PAEC to 15 µM NO constitutes a pathophysiologically relevant cellular model with which to study NO-mediated persistent inhibition of complex IV in vascular endothelial cells. Cultured cells were exposed to 01 mM NOC-18 for 024 h. Some NO-treated cells were rinsed three times and incubated in fresh medium for 6 h. Persistent inhibition of complex IV was defined in this study as activity loss after 6-h incubation in NO-free medium.
Affinity chromatography purification of complex IV. Mitochondrial particles were prepared from porcine PAEC as previously described (69). The particles were subjected to affinity chromatography as previously described (12, 65). Briefly, the mitochondrial fractions were suspended in 100 mM potassium phosphate buffer, pH 7.8, and extensively dialyzed against 25 mM sodium bicine, pH 7.9. The mitochondrial fractions (20 mg protein/ml) were made, and 2 mg of lauryl maltoside was added per milligram of protein. The solution was stirred for 30 min and then centrifuged at 40,000 g for 30 min. The supernatant was diluted to 1.5 mg of protein/ml in 0.75% lauryl maltoside. A horse cytochrome c-CNBr-Sepharose 4B column (Amersham Biosciences) was prepared, reduced with 5 mM ascorbate, and equilibrated on the bench top in cold chromatography buffer: 25 mM sodium bicine, pH 7.9, 15 mM lauryl maltoside, and 5% sucrose. The proteins were loaded on the column, washed with 3 column volumes of the chromatography buffer, and eluted by the addition of 4 column volumes of linear gradient of 00.2 M NaCl. The protein and complex IV contents were monitored by measurements of absorbances at 280 and 420 nm, respectively. The eluted complex IV is spectrally pure, with an average heme-to-protein ratio of 11.913.0 nmol heme a/mg of protein and a very low 280 nm-to-420 nm absorbance ratio of 1.8.
Measurement of complex IV activity.
Mitochondrial protein or purified complex IV was used to measure complex IV activity by following the oxidation of reduced cytochrome c at 550 nm with extinction coefficient 550 = 27.7 mM1cm1 as previously described (66, 69). The specific activity of complex IV was expressed as micromoles per minute per milligram of protein, and then relative activities (% of activity in control cells) for 424 h time points were calculated.
Measurement of sulfhydryls of complex IV.
The effects of NO on free sulfhydryl reactivity were determined by titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by the method of Ellman and Gan (25) and Riddles et al. (51) under denaturing conditions. After exposure to NO (NOC-18, 1 mM) or medium only for 024 h and then fresh medium for 6 h, PAEC were collected for isolation of mitochondria with a mitochondrial fraction kit (Active Motif, Carlsbad, CA; Refs. 69, 71). Complex IV purified from the mitochondrial fraction was dialyzed against PBS, and protein (5 µM) was denatured with 6 M guanidine HCl and reacted with DTNB (200 µM) in PBS at room temperature for 90 min. The reaction was monitored as an increase in absorbance at 412 nm. The concentration of SH groups was calculated with 412 = 13,700 M1cm1 (51), and the results are expressed as number of SH groups per complex IV monomer. In some experiments, 50100 µM carboxyl-PTIO was added 5 min before NO exposure and remained present throughout the 24-h exposure.
Assessments of GSH-to-GSSG ratios in mitochondria. Mitochondrial fractions were isolated from PAEC with or without treatment with 1 mM NOC-18 for 18 h as previously described (69, 71). Mitochondrial GSH-to-GSSG ratios were assessed with the GSH/GSSG Ratio Assay kit (Calbiochem; Refs. 69, 71). Briefly, 10 µl of 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP), a thiol-scavenging reagent to rapidly scavenge GSH, was added to 100 µl of mitochondrial fraction, which was used for GSSG sample preparation. For GSH sample preparation, 50-µl mitochondrial fractions without the presence of M2VP were used. The mixture of sample-blank-standard, chromogen, enzyme, and NADPH (200 µl of each) in a cuvette was examined for the change of absorbance at 412 nm for 3 min with a spectrophotometer. The reaction rate and calibration curves were used to calculate concentrations of GSH and GSSG. GSH-to-GSSG ratios were then calculated.
Adenovirus-mediated allotopic expression of Trx in mitochondria and attenuation of NO inhibition of complex IV. The Trx gene was rescued from pcDNA3-Trx (70) and cloned into pCMV/myc/mito vector, a vector for allotopic expression, i.e., targeting proteins to mitochondria with a signal sequence encoding a leader peptide to import expressed protein into mitochondria and a myc epitope fused to the COOH terminal for detection of allotopic expression in mitochondria (Invitrogen). The Trx cDNA with additional mitochondrial signaling sequence (coming from the pCMV/myc/mito vector) was rescued and cloned into a transfer vector, pShuttle-CMV, to form pAd-Trx. The same vector containing a green fluorescent protein (GFP) gene, pAd-GFP, and the sham vector, pAd, were made for controls. After sequences were verified by restriction enzyme digestion and sequencing [DNA Sequencing Core, Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida], the assembled transfer vector was transferred into the adenovirus (Ad) genome by homologous recombination. The recombinant Ad constructs were then cleaved with PacI to expose its inverted terminal repeats and transfected into QBI-293A cells to produce viral particles Ad-Trx, Ad-GFP, and Ad, respectively. The viruses Ad-Trx, Ad-GFP, and Ad [5 x 108 plaque-forming units (pfu)/ml] were used to infect PAEC (95% confluent) by incubating at 37°C for 48 h. The specific Trx gene expression in mitochondria of Ad-infected PAEC was confirmed by Western blot analyses of Trx in mitochondrial and cytosolic fractions with an anti-Trx antibody (American Diagnostics) as described previously (69, 71, 73). Relative levels of Trx in the mitochondrial fraction isolated from Ad-Trx-infected cells were elevated two times compared with Ad-Trx-infected cells, indicating an overexpression of Trx in mitochondria. The Ad-Trx- or Ad-GFP-infected cells were exposed to 1 mM NOC-18 or control medium for 18 h and then fresh medium for 6 h. The treated cells were harvested and analyzed for complex IV activity.
Identification of putative NO-sensitive motifs in complex IV by sequence analysis. Complex IV in mammalian mitochondria consists of at least 15 subunits, subunits I, II and III, IVa, IVb, Va, Vb, VIa, VIb, VIIa, VIIb, VIIc, VIIIa, VIIIb, and VIIIc (30). Peptide sequences of complex IV subunits available in the Swiss-Prot database were compared to identify highly conserved regions that contain cysteines and residues that enhance NO-cysteine reactivity, e.g., arginine, aspartate, glutamate, histidine, and lysine. We determined 1) whether cysteine residues are present in the peptide of complex IV subunits, 2) whether cysteine residues are highly conserved across all species, and 3) whether a putative NO-sensitive conformation exists.
Immunoprecipitation and Western blot analysis of nitrosylated complex IV S2. Proteins were immunoprecipitated from NO-treated (01 mM NOC-18 for 18 h, then fresh medium for 6 h) or control (medium only) PAEC with an anti-nitrosocysteine antibody (4 µg, mouse monoclonal; A. G. Scientific) or 4 µg of isotype-matched control antibody in the dark. The antigen-antibody complexes were isolated with protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4°C. The beads were then washed five times in high-salt buffer to which 1 mM N-ethylmaleimide was added to block free thiols and thereby prevent artifactual S-nitrosylation. Boiled immunoprecipitates in loading buffer with or without DTT were loaded on a 15% SDS-PAGE gel, blotted on nitrocellulose membranes, and hybridized to an anti-complex IV S2 antibody (1:400 dilution, mouse monoclonal; Molecular Probes) and a secondary antibody (anti-mouse IgG conjugated to horseradish peroxidase; Molecular Probes). The protein bands were visualized with enhanced chemiluminescence detection reagent (Amersham) and Bio-Max X-ray film. Band intensities were examined on a densitometer (Fluor-S MultiImager System; Bio-Rad).
"Biotin switch" assay of nitrosylated complex IV S2. Biotin switch assay was performed as described previously with slight modifications (33, 41). In brief, control- or NO-exposed PAEC (1 mM NOC-18 for 18 h, then fresh medium for 6 h) were incubated in a nondenaturing lysis solution (in mM: 50 Tris·HCl, pH 7.4, 300 NaCl, 5 EDTA, and 0.1 neocuproine with 1% Triton X-100, aprotinin, and leupeptin). Free thiols were blocked by incubation of the samples in the blocking buffer [in mM: 225 HEPES, pH 7.7, 0.9 EDTA, 0.09 neocuproine, and 20 methylmethanethiosulfonate (MMTS) with 2.5% SDS, 20 min at 50°C]. MMTS was removed by protein precipitation with acetone. The pellet was resuspended in HENS buffer (in mM: 250 HEPES, pH 7.7, 1 EDTA, and 0.1 neocuproine with 1% SDS). N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio) propionamide (biotin-HPDP, 0.4 mM; Amersham Biotech) and sodium ascorbate (1 mM) were added, and the mixture was incubated for 1 h at room temperature in the dark to replace the NO group with biotin on the thiols of cysteine residues. After acetone precipitation, the biotin-labeled proteins were purified with neutravidin-agarose (15 µl/mg protein, Amersham Biotech). Biotinylated proteins were separated from the agarose beads by boiling in the SDS-PAGE gel loading buffer and subjected to Western blot analysis using anti-complex IV S2 antibody to detect biotinylated complex IV S2 (see above).
Site-directed mutagenesis of complex IV S2. Cys196 and Cys200 residues of complex S2 were mutated to Ala by changing the Cys codon to Ala codon in the S2 cDNA as described previously (29, 74). The S2 cDNA was modified for nuclear expression, i.e., four TGA codons (for Trp in mitochondria, but for stop in cytosol) were mutated to TGG codons (for Trp in cytosol). The cDNAs with the mutated codons (Cys196/Cys200 to Ala196/Ala200) were then amplified with the modified cDNA of complex IV S2 as a template and primers 1 and 2/3. Sequences of primers 1 and 2/3 were 5'-GTC GAC ATG GCT TAC CCT TTC CAA CTA GGC TTC-3' and 5'-GCGGCCGC TTA ACC TGT TAA TAT TGA TGT TGA CCA TTT TTC GAA GTA CTT TAA TGG GAC AAG TTC AAG TAC AAT GGG CAT GAA GCT GTG GTT TGA TCC GGC GAT TTC TGA GGC-3', which contain SalI (in primer 1) and NotI (in primer 2/3) restriction sites (underlined) and the changed codons for Ala196 (bold) (in primer 2) and Ala200 (bold) (in primer 3) from Cys196 and Cys200. The mutated cDNA was cloned into pCMV/myc/mito vector in frame between sites for SalI and NotI to form pS2196 and pS2200, respectively. The mutations from Cys to Ala codon of pS2196 and pS2200 were confirmed by nucleotide sequencing (DNA Sequencing Core, ICBR, University of Florida). The mutated S2 cDNA with additional mitochondrial signaling sequence was cloned into pShuttle-CMV to form pAd-S2196 or pAd-S2200. After sequences were verified, the assembled transfer vector was transferred into the Ad genome. Viral particles Ad-S2196, Ad-S2200, Ad-GFP, and Ad were produced.
Allotopic expression of mutated complex IV S2. The viruses Ad-S2196, Ad-S2200, Ad-GFP, and Ad (5 x 108 pfu/ml) were used to infect PAEC (95% confluent) by incubating at 37°C for 48 h. Allotopic expression of mutated complex IV S2 was verified by Western blot analysis of complex IV S2 in the mitochondrial fraction of the infected cells with antibodies against myc tag for mutants and complex IV S2 for total S2 protein. The infected PAEC were exposed to 1 mM NOC-18 or control medium for 18 h and then incubated in fresh medium for 6 h. These cells were harvested and assessed for S2 nitrosylation and complex IV activity.
Statistical analysis. Significance for the effect of NO-induced loss of complex IV activity and the protective effects of Trx was determined by analysis of variance and Student's t-test (68).
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RESULTS |
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NO-induced S-nitrosylation of complex IV S2. To determine whether exposure of cells to NO causes nitrosylation of cysteine residues on complex IV S2, two approaches were used, namely, immunoprecipitation and Western blot analysis and the biotin switch assay. After exposure of PAEC to 1 mM NOC-18 or control medium for 18 h and then to fresh medium for 6 h, S-nitrosylated proteins were immunoprecipitated from cell lysates with an anti-nitrosocysteine monoclonal antibody. Western blotting analysis of S-nitrosylated complex IV S2 in the immunoprecipitates was carried out with an anti-complex IV S2 antibody (Fig. 5A). Band intensities of nitroso-complex IV S2 were determined and plotted (Fig. 5B). As shown in Fig. 5, A and B, the level of S-nitrosylated complex IV S2 protein in NO-exposed cells is two times higher than that in the control cells.
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NO-induced S-nitrosylation of complex S2 is dose dependent. PAEC were exposed to 01 mM NOC-18, equivalent to 03 µM NO in medium, for 18 h and then incubated in fresh medium for 6 h at 37°C. Cells were collected and used for immunoprecipitation and Western blot analysis of S-nitrosylated complex IV S2. The content of nitrosylated complex IV S2 in cells exposed to low levels of exogenous NO (200 nM NO released from 0.25 mM NOC-18) is comparable to that in control cells (Fig. 6). Exposure of PAEC to high levels of NO (>1 µM NO released from 0.751 mM NOC-18) increased S-nitrosylation of complex IV S2 twofold.
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DISCUSSION |
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Sequence analysis of complex IV peptides revealed a novel putative NO-sensitive motif, Gln-Cys196-Ser-Glu198-Ile-Cys200-Gly, in complex IV S2. Mammalian complex IV consists of at least 15 subunits (30), and cysteine residues in all subunits can be potential targets for NO modification. However, target cysteine residues must be accessible and sensitive to NO and must be essential active site residues of complex IV if modification of the cysteines is to serve as an initiation factor for NO signaling. The two cysteines in complex IV S2 are located in the active center that converts O2 to H2O; hence, they are as accessible to NO as to O2 (1, 55). On the basis of the human Hb structure and accounting for the known acid-base catalyzed Cys 93 nitrosylation and Cys
393 NO-denitrosylation processes, a putative amino acid sequence, (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu), has been proposed as the minimum consensus motif for Cys-NO reactivity (5). The most important component of the tri- or tetrapeptide consensus motif has been recognized as the Cys(Asp/Glu) pair (59). An acidic residue, Glu198, in the putative motif Gln-Cys196-Ser-Glu198-Ile-Cys200-Gly of complex IV S2 may enhance the interaction between the two cysteines and NO, which makes the two cysteines more sensitive to NO, i.e., the two cysteines are more likely modified by NO than other cysteines. NO may react with O2 to form N2O3 in the active site of complex IV, which can immediately attack the nearby cysteine residues of complex IV S2. This selective S-nitrosylation can lead to inhibition of complex IV activity. An autocatalytic mechanism of protein nitrosylation may explain why only particular cysteine residues are targeted within a protein and other cysteine residues are left unmodified (46). These NO-sensitive cysteine residues are essential to bridge two copper atoms to form a Cu A center (10, 30). NO-induced oxidation and S-nitrosylation of complex IV S2 may prevent the copper center repair, releasing copper ions. The free copper may catalyze tyrosine nitration of complex IV S2, because Thomas et al. (64) demonstrated that NO-induced protein nitration can be mediated through free heme and metals. Nitration of complex IV can result in its degradation and loss of activity. In addition, the two cysteines in the putative motif are highly conserved across species, which also suggests a vital role for these cysteines in maintaining integrity of complex IV. Similar conformations have been found in the two-cysteine nuclear factors, NF-
B (22, 23) and AP-1 (62). Redox regulation of the two cysteines in these nuclear factors modulates their binding activities.
Our experimental results support the notion that NO oxidizes/nitrosylates the two cysteines in the putative NO-sensitive motif. For instance, exposure of PAEC to NO increased S-nitrosylation of complex IV S2 as determined by two methods, namely immunoprecipitation and Western blot analysis and biotin switch assay, and the effects of NO on nitrosylation were dose dependent. There are only two cysteine residues in the peptide of complex IV S2, which are located in the putative NO-sensitive motif; therefore, NO-increased nitrosylation of complex IV S2 indicates nitrosylation of one or both of the two critical cysteines. Furthermore, removal of thiol groups via replacement of the cysteines by alanines with site-directed mutagenesis diminished NO-induced S2 nitrosylation, verifying the reaction between NO and these key cysteines of complex IV S2. Because the thiol groups of S2 are essential for complex IV activity, removal of the thiols is expected to cause loss of complex IV activity, which was observed in our mutagenesis studies of complex IV S2.
We provide evidence here of a causal relationship between NO-induced persistent inhibition of complex IV and oxidation/S-nitrosylation of cysteine residues located in a putative NO-sensitive motif of complex IV S2 in porcine PAEC. The hypothesis is put forth that NO modulation of complex IV via cysteine oxidation/nitrosylation plays a critical role in NO-induced cytotoxicity of vascular endothelial cells via mitochondrion-mediated pathways. Lung endothelium is exposed to multiple NO sources under pathological conditions, e.g., NO generated by endogenous NOS, NO inhalation therapy, NO in polluted air or smoke, and NO produced by inducible NOS in inflammatory cells (2, 31, 32, 34, 37, 67). Despite its physiological importance, excessive NO has cytotoxic effects on vascular endothelial cells, e.g., oxidative injury and apoptosis (4, 21, 24, 28, 44, 45, 54, 61). Therefore, patients receiving long-term NO inhalation therapy may have impaired endothelial function due to oxidative injury and cell death. Similar loss of lung endothelial function can occur in the event that excessive NO is generated by endogenous processes as a result of diverse pulmonary disorders and/or inflammatory conditions or generated by environmental exposure (20, 24, 27, 37, 38, 57). Therefore, identification of the novel molecular mechanisms underlying NO inhibition, particularly persistent inhibition, of complex IV via oxidation/nitrosylation of critical cysteine residues can help us better understand NO-induced mitochondrial and endothelial dysfunction under pathological conditions.
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ACKNOWLEDGMENTS |
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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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