Induction of Reversible Cysteine-Targeted Protein Oxidation by an

We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-Δ12,14-prostaglandin. J2 (15d-PGJ2), is the potent inducer of intracellular oxi...
0 downloads 0 Views 280KB Size
Download PDF
Chem. Res. Toxicol. 2004, 17, 1313-1322

1313

Induction of Reversible Cysteine-Targeted Protein Oxidation by an Endogenous Electrophile 15-Deoxy-∆12,14-prostaglandin J2 Takeshi Ishii† and Koji Uchida*,†,‡ Graduate School of Bioagricultural Sciences and Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan Received May 18, 2004

We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J. Biol. Chem. 276, 12076-12083.]. In the present study, to investigate the correlation between the redox regulation and the 15d-PGJ2-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins using biotinylated cysteine as a molecular probe. In addition, the reversible regulation of protein function by S-oxidation/thiolation was characterized in vitro. When human neuroblastoma SHSY5Y cells were exposed to 15d-PGJ2, followed by treatment with biotinylated cysteine, 26 proteins, including glycolytic enzymes, cytoskeletal proteins, redox enzymes, and stress proteins, were identified as substrates for reversible cysteine-targeted oxidation. To investigate the regulatory mechanism of protein function by S-oxidation/thiolation, the binding of a low molecular weight thiol (glutathione) to a glycolytic enzyme R-enolase was characterized. Treatment of R-enolase with the thiol oxidant diamide in the presence of glutathione in vitro resulted in the binding of glutathione to the protein and concomitant loss of the enzymatic activity, whereas the glutathiolation and inactivation of R-enolase were fully reversed by dithiothreitol. Mass spectrometric analysis of the tryptic fragments from native and oxidized R-enolase identified two cysteine residues, Cys-118 and Cys-388, as the S-oxidation sites, which may play a role in the regulation of the biological activities of the protein and may be regulated by a reversible S-oxidation/thiolation reaction. These results suggest that cysteine-targeted oxidation/thiolation plays a critical role in the regulation of protein function under conditions of electrophile-induced oxidative stress.

Introduction Oxidative stress is increasingly seen as a major upstream component in the signaling cascade involved in many of the cellular functions, such as cell proliferation, inflammatory responses, stimulating adhesion molecule, and chemoattractant production (1). It has been suggested that some level of oxidative stress may be required in response to cytotoxic agents and converted into the redox regulatory system as a downstream signaling pathway (2). However, excess oxidative stress may be toxic, exerting cytostatic effects, causing membrane damage, and activating pathways of cell death (apoptosis and/ or necrosis). Oxidative stress has the dual effects of causing damage, as well as initiating stress adaptive or protective responses. The prostaglandins (PGs) are a family of structurally related molecules that are produced by cells in response to a variety of extrinsic stimuli and regulate cellular growth, differentiation, and homeostasis. PGs are derived from fatty acids, primarily arachidonate, which are * To whom correspondence should be addressed. Fax: 81-52-7895741. E-mail: [email protected]. † Graduate School of Bioagricultural Sciences. ‡ Institute for Advanced Research.

released from membrane phospholipids by the action of phospholipases. Arachidonate is first converted to an unstable endoperoxide intermediate by cyclooxygenase and subsequently converted into one of several related products, including PGD2, PGE2, PGF2R, prostacyclin (PGI2), and thromboxane A2, through the action of specific PG synthetases. Among them, PGD2 is a major cyclooxygenase product in a variety of tissues and cells and has marked effects on a number of biological processes, including platelet aggregation, relaxation of vascular and nonvascular smooth muscles, and nerve cell functions (3). It has been shown that PGD2 readily undergoes dehydration in vivo and in vitro to yield biologically active PGs of the J2 series, such as PGJ2, ∆12,14-PGJ2, and 15-deoxy∆12,14-PGJ2 (15d-PGJ2)1 (4-7). Members of the J2 series of the PGs, unlike other classes of eicosanoids, characterized by the presence of an electrophilic R,β-unsaturated carbonyl group in the cyclopentenone ring, have their own unique spectrum of biological effects, including inhibition of macrophage-derived cytokine production (8, 1 Abbreviations: 15d-PGJ , 15-deoxy-∆12,14-PGJ ; GSH, glutathione, 2 2 reduced form; GSSG, glutathione, oxidized form; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; HRP, horseradish peroxidase; PBS, phosphate buffered saline; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

10.1021/tx049860+ CCC: $27.50 © 2004 American Chemical Society Published on Web 09/30/2004

1314

Chem. Res. Toxicol., Vol. 17, No. 10, 2004

9) and IκB kinase (10, 11), induction of synoviocyte and endothelial cell apoptosis (12), induction of glutathione (GSH) S-transferase gene expression (13), and potentiation of apoptosis in neuronal cells (14). Moreover, recent studies have shown that 15d-PGJ2 directly inhibits the NF-κB-dependent gene expression through covalent modification of critical cysteine residues in IκB kinase (10) and the DNA-binding domains of NF-κB subunits (11, 15). In our previous study, we demonstrated for the first time that 15d-PGJ2 was accumulated in the spinal cord of sporadic amyotrophic lateral sclerosis patients, mainly occurring in the motor neurons of the anterior horn (16). This finding raised the possibility that cyclooxygenase-2 up-regulation, through its pivotal role in inflammation, followed by the enhanced intracellular production of 15dPGJ2, might be ubiquitously involved in neurodegenerative processes. On the other hand, as part of an effort to identify the endogenous inducer of intracellular oxidative stress and to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we examined the oxidized fatty acid metabolites for their ability to induce intracellular reactive oxygen species (ROS) production in SH-SY5Y cells in vitro and found that the J2 series of the PGs represent the most potent inducers (17). On the basis of the observations that (i) 15d-PGJ2 partially reduced intracellular GSH levels, (ii) 15d-PGJ2 treatment of the cells resulted in a significant decrease in the GSH peroxidase activity, and (iii) the N-acetylcysteine pretreatment significantly inhibited both ROS production and cytotoxicity by 15d-PGJ2, the intracellular redox status appeared to represent a critical parameter for the PG-induced ROS production and cytotoxicity. Furthermore, it was observed that the intracellular ROS production was accompanied by the alteration of the cellular redox status and the production of lipid peroxidation-derived highly cytotoxic aldehydes, such as acrolein and 4-hydroxy-2-nonenal (17). On the basis of these findings, we have hypothesized that intracellular oxidative stress constitutes a pivotal step in the pathway of cellular dysfunction induced by electrophilic molecules. In the present study, to investigate the correlation between the redox regulation and the 15dPGJ2-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins generated in response to intracellular oxidative stress induced by 15d-PGJ2. In addition, the mechanism for cysteinetargeted oxidation/thiolation of a glycolytic enzyme was characterized.

Experimental Procedures Materials. 15d-PGJ2 was purchased from the Cayman Chemical Co. (Ann Arbor, MI). Biotinylated cysteine (biotincysteine) and GSH (biotin-GSH) were prepared as previously described (18, 19). Horseradish peroxidase (HRP)-conjugated NeutrAvidin, Cy5-labeled avidin, and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Biosciences. The protein concentration was measured using the BCA protein assay reagent obtained from Pierce. L-Cysteine and R-enolase were from Sigma. The SYPRO Ruby protein gel stain was from Molecular Probes, Inc. Sequence grade modified trypsin was obtained from Promega. Dithiothreitol (DTT) and iodoacetoamide were obtained from Wako Pure Chemical Industries, Ltd. Cell Culture. SH-SY5Y cells were grown in Cosmedium-001 (Cosmo-Bio, Tokyo) containing 5% Nakashibetsu precolostrum

Ishii and Uchida newborn calf serum, 100 µg/mL penicillin, and 100 units/mL streptomycin. The cells were seeded in plates coated with polylysine and cultured at 37 °C. Preparation of S-Oxidized/Thiolated Cellular Proteins. The cells were incubated with 10 µM 15d-PGJ2 in a control medium for 1 h. The medium was removed and replaced with the same volume of control medium containing 100 µM biotincysteine for 15 min. The cells were then washed twice with phosphate-buffered saline (PBS), lysed in lysis buffer [4% (w/v) CHAPS, 9 M urea, 40 mM Tris (base)], and centrifuged at 10000 rpm for 5 min at 4 °C; the supernatant was assayed for protein content and then stored at -80 °C until use. Immunocytochemistry. The cells were fixed overnight in PBS containing 2% paraformaldehyde and 0.2% picric acid at 4 °C. The membranes were permeabilized by exposing the fixed cells to PBS containing 0.3% Triton X-100. The cells were then sequentially incubated in PBS solutions containing blocking serum (4% normal goat serum) and immunostained with Cy5labeled avidin, rinsed with PBS containing 0.3% Triton X-100, and covered with anti-fade solution. Images of cellular immunofluorescence were acquired using a confocal laser microscope (Bio-Rad) with a 40× objective (488 nm excitation and 518 nm emission). Western Blot Analysis. For detection of the S-oxidized proteins, whole cell lysates from SH-SY5Y cells were treated with biotin-cysteine and then incubated with sodium dodecyl sulfate (SDS) sample buffer with or without a reducing agent for 5 min at 100 °C. The samples were separated by 10% SDSpolyacrylamide gel electrophoresis (PAGE). The gel was transblotted onto a nitrocellulose or PVDF membrane, incubated with Blockace for blocking, washed, and incubated with the HRPNeutrAvidin. This procedure was followed by the addition of ECL reagents. The bands were visualized by Cool Saver AE6955 (ATTO, Tokyo, Japan). Protein Separation by One-Dimensional and TwoDimensional Electrophoresis. For the identification of Soxidized proteins by one-dimensional electrophoresis, the total cell lysate (100 µg) treated with or without 50 mM DTT was heated at 95 °C for 5 min and resolved by SDS-PAGE, and the proteins were transferred to the nitrocellulose membrane for Western blot analysis. For the identification of S-oxidized proteins by two-dimensional electrophoresis, samples containing 300 µg of total cell lysate [supplemented with 1% immobilized pH gradient (IPG) buffer, pH 3-10] were used to rehydrate IPG strips, pH 3-10 (Amersham Biosciences, Inc.). First dimension electrophoresis was performed using the following program: 1 h at 500 V, 1 h at 1000 V, and 6 h at 8000 V. Prior to second dimension electrophoresis, IPG strips were equilibrated for 20 min in 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, and 0.01% bromphenol blue without reducing and alkylating agent. The second dimension was performed on a 10% gel at a constant 25 mA per gel. Separated proteins were then fixed in the gel using 40% ethanol and 10% formic acid, stained with SYPRO Ruby protein gel stain, and scanned using the Typhoon 9400 (Amersham Biosciences, Inc.). Western blot analyses were previously described. The protein spots were visualized by Image Quant (Amersham Biosciences, Inc.). Identification of S-Oxidized Proteins. Gel pieces were washed in water containing 0.1 and 50% methanol for 1 h, dehydrated in acetonitrile, and dried in a SpeedVac for 30 min. The samples were proteolyzed with 1 µg of sequence grademodified trypsin in 100 mM Tris-HCl buffer (pH 8.8) overnight at 37 °C. The supernatant was collected, and the peptides were further extracted with 50% acetonitrile containing 0.1% trifluoroacetic acid. The peptide extracts were vacuum-dried and resuspended in 50% acetonitrile containing 0.1% formic acid. Peptide mass fingerprints were generated with an AutoFLEX matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometer (Bruker Daltonics Japan, Ltd., Tokyo, Japan). A few microliters of the sample were mixed with an equal volume of a saturated solution of sinapinic acid or R-ciano-

Targets of Oxidative Stress Induced by 15d-PGJ2 4-hydroxycinnamic acid (Sigma) in acetonitrile/0.1% trifluoroacetic acid 1:3 (vol/vol); 1 µL of the mixture was deposited on the MALDI-TOF mass spectrometry target. The proteins were identified with the MASCOT (Matrix Science, London) searching algorithms using the nonredundant database. Probability-based MOWSE scores were estimated by comparison of search results against estimated random match population and were reported as ∼10*LOG10(p), where p is the absolute probability. Scores greater than 63 were considered significant, meaning that for scores higher than 63 the probability that the match is a random event is lower than 0.05. All protein identifications were in the expected size and PI range based on the position in the gel. Inactivation of r-Enolase by Diamide and GSH. R-Enolase (0.3 mg) was incubated with an increasing amount of diamide (1 mM) or diamide plus GSH (1 mM) in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. The reaction was terminated by centrifugal filtration (Microcon 30, molecular weight cutoff of 30000) to remove the low molecular weight reactants. An aliquot of the sample was then removed and analyzed for activity. The enolase activity was measured essentially as previously described (20). Attempts to reverse the effect of diamide plus GSH on enolase were carried out by adding 10 mM DTT to filtered enzyme samples after treatment with 1 mM diamide plus 1 mM GSH, and incubation was continued for an additional 30 min at 25 °C before assay. Enzyme-Linked Immunosorbent Assay (ELISA). R-Enolase (0.3 mg) was incubated with an increasing amount of biotinGSH (0-2 mM) plus diamide (0-2 mM) in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. The reaction was terminated by centrifugal filtration to remove the low molecular weight reactants. A 100 µL (100 µg/mL) aliquot of the antigen solution was added to each well of a 96 well microtiter plate and incubated overnight at 4 °C. The antigen solution was then removed, and the plate was washed with PBS containing 0.05% Tween 20 (PBS/Tween) and water. Each well was filled with Block Ace solution (40 mg/mL) for 1 h at 37 °C. The HRPNeutrAvidin was then added to the wells at 100 µL/well of 1 mg/mL solution for 2 h at 37 °C. After the supernatants were discarded and washed three times with PBS/Tween, the enzymelinked NeutrAvidin bound to the well was revealed by adding 100 µL/well of 1,2-phenylenediamine (0.5 mg/mL) in 0.1 M citrate/phosphate buffer (pH 5.0) containing 0.003% H2O2. The reaction was terminated by the addition of 50 µL of 2 M sulfuric acid, and the absorbance at 492 nm was read on an Ultramark microplate imaging system (Bio-Rad). Identification of Target Cysteines in r-Enolase. After the treatment of R-enolase samples with diamide, biotin-GSH, or diamide plus biotin-GSH as described, an aliquot of the sample was added to the sample buffer with or without a reducing agent for 5 min at 100 °C. Samples (3 µg/well) were then separated by 10% SDS-PAGE. The gels were then stained with SYPRO Red protein gel stain and transblotted onto a nitrocellulose membrane. Immunoblot analyses were carried out as previously described. Native and S-thiolated enolase were mixed with a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid containing 75% acetonitrile and 0.1% trifluoroacetic acid and dried on stainless steel targets at room temperature and pressure. The analyses were performed using an AutoFLEX MALDI-TOF mass spectrometer (Bruker Daltonics Japan, Ltd.) with a nitrogen laser (337 nm). All analyses were in the positive ion mode, and the instrument was calibrated immediately prior to each series of studies.

Results Induction of Cysteine-Targeted Protein Oxidation by 15d-PGJ2. To detect cellular proteins that undergo cysteine-targeted oxidation during the electrophile-induced oxidative stress, biotin-cysteine was utilized as a molecular probe (Figure 1A). This probe rapidly crosses the plasma membrane and can be used to detect, quantify, purify, and identify proteins susceptible to

Chem. Res. Toxicol., Vol. 17, No. 10, 2004 1315

Figure 1. Formation of S-oxidized proteins in SH-SY5Y cells exposed to 15d-PGJ2. (A) Chemical structure of biotin-cysteine. (B) Immunocytochemical detection of biotin-cysteine-protein adducts. SH-SY5Y cells were incubated with 10 µM 15d-PGJ2 for 1 h and then treated with biotin-cysteine (0-100 µM) for 15 min. (C) Western blot analysis of biotin-cysteine-protein adducts. Cells were incubated with 10 µM 15d-PGJ2 for 1 h and then treated with 100 µM biotin-cysteine for 15 min. Total cell lysate (100 µg) treated with or without 50 mM DTT was heated at 95 °C for 5 min and resolved by SDS-PAGE followed by Western blot analysis. The differences in the signals among the various protein bands from those samples treated with or without 50 mM DTT reflected the extent of biotin-cysteine binding.

oxidation in all compartments of the cell (18). As shown in Figure 1B, when SH-SY5Y cells were treated with 10 µM 15d-PGJ2 for 1 h followed by treatment with biotincysteine (0-100 µM), significant incorporation of biotincysteine into the cells was observed. To determine the number and range of proteins that are S-oxidized, the cells exposed to 15d-PGJ2 were treated with biotincysteine, and the biotin-cysteine-protein conjugates were analyzed by Western blot analysis probed with streptavidin-HRP. As shown in Figure 1C (left), several un-

1316

Chem. Res. Toxicol., Vol. 17, No. 10, 2004

Ishii and Uchida

Figure 2. Two-dimensional gel electrophoresis of S-oxidized proteins. SH-SY5Y cells were incubated with 10 µM 15d-PGJ2 for 1 h and then treated with 100 µM biotin-cysteine for 15 min. The proteins were separated by isoelectrofocusing (pH range 3-10) and then by SDS-PAGE. All runs were carried out under nonreducing conditions. (A,C) SYPRO Ruby fluorescence staining. (B,D) Western blot. The red arrowheads denote spots excised for subsequent identification by MALDI-TOF analysis, as described under Experimental Procedures.

known proteins were found to be S-oxidized in response to 15d-PGJ2 (e.g., of 37, 47, 56, 70, and 90 kDa). Hydroperoxides, such as H2O2 and tert-butylhydroperoxide, induced a similar pattern of cysteine-targeted oxidation (data not shown). The treatment of samples with DTT almost completely abolished the signal (Figure 1C, right), demonstrating that the signals are detected as a consequence of a disulfide bond linking the biotincysteine to a protein. There are two dominant protein bands that are not reducible and are also present in the control tissue. These are either proteins that nonspecifically bind streptavidin-HRP, or they are proteins that are endogenously biotinylated in the cells. Identification of S-Oxidized Proteins. To identify S-oxidized proteins in the cells exposed to 15d-PGJ2, the biotin-cysteine-protein conjugates were separated on two-dimensional gel electrophoresis and analyzed by Western blot analysis probed with streptavidin-HRP. As shown in Figure 2, several S-oxidized proteins were detected following 15d-PGJ2 treatment. Spots were then excised from two-dimensional gels, subjected to trypsin digestion, and then successfully analyzed by MALDI-TOF mass fingerprint analysis. The multiple gel spots for a single identification may be due to post-translational modification, such as phosphorylation. However, at this moment, it is impossible by visual inspection of a pattern of spots on a gel to determine which modifications are most likely to be present. Figure 3 shows mass spectra

Figure 3. Identification of R-enolase as the target of cysteinetargeted protein oxidation. MALDI-TOF MS spectrum corresponding to R-enolase tryptic digest. The m/z values obtained from tryptic peptides were used to identify the protein by searching in a database.

corresponding to a 47 kDa S-oxidized protein (spot no. 21) shown in Figure 2. Using MASCOT, the probabilitybased MOWSE score was 235 for R-enolase (p < 0.05), with 19 peptide matches (error (0.01%), which represents 44% sequence coverage (Supporting Information). Table 1 lists the identity of 49 protein spots, which could be identified in one or more of three independent experiments. The identified proteins fall into several different functional classes, including cytoskeletal proteins (tropomyosin, tublin, and actin), glycolytic enzymes (enolase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, and

Targets of Oxidative Stress Induced by 15d-PGJ2

Chem. Res. Toxicol., Vol. 17, No. 10, 2004 1317

Table 1. Summary of Oxidation Sensitive Proteins Identified spot no.

protein name

Mw

PI

1 2, 3 4 5, 6 7 8, 9 10 11, 12 13 14 15-17 18 19-22 23, 24 25, 26 27 28, 29 30-34 35-37 38 39 40 41-43 44 45-47 48

heat shock protein gp96 precusor (tumor rejection antigen 1) heat shock protein 90 kDa-R heat shock protein 70 kDa protein 5 (glucose-regulated protein 78 kDa) heat shock protein 70 kDa protein 8 isoform 1 prolyl 4-hydroxylase, β-subunit, β-subunit protein disulfide-isomelase ER60 precusor tublin R 6 or 2 translation elongation facter EF-Tu precursor γ, enolase (enolase 2 neuronal) proteasome 26S ATPase subunit 4 isoform 1 protein disulfide isomerase-related protein 5 γ, enolase (enolase 2 neuronal) R-enolase aldolase actin, β unr interacting protein lactate dehydrogenase B glyceraldehyde 3-phosphate dehydrogenase heaterogenous nuclear ribonucleoprotein A2/B1 isoform tyrosin 3 tryptophan 5 monooxigenase activated protein nucleophosmin/B23.2 hepatoma-derived growth factor heat shock protein 27 kDa ubiquitin calboxy-terminal esterase L1 GSH-S-transferase Pi-1 peroxiredoxin 2 isoform a

90138 84621 72288 70854 57480 56761 49863 49843 47581 47337 46170 47581 47139 39420 41321 38428 36615 36031 35984 29155 28383 26772 22760 24808 23367 21878

4.73 4.94 5.09 5.37 4.76 5.98 4.96 7.26 4.91 5.03 4.95 4.91 7.1 8.3 5.56 4.99 5.71 8.26 8.67 4.63 4.86 4.9 5.98 5.3 5.43 5.66

lactate dehydrogenase), redox enzymes (peroxiredoxin and GSH-S-transferase), proteasome subunit, prolyl 4-hydroxylase, ubiquitin calboxy-terminal esterase, chaperone protein (HSP27, HSP70, HSP90, and protein disulfide isomelase), hepatoma-derived growth factor, translation elongation facter, cyclophilin, nuclear ribonucleoprotein, nucleophosmin, and unr interacting protein. Binding of Biotin-Cysteine to Endogenous r-Enolase during the 15d-PGJ2-Induced Oxidative Stress. To characterize the regulatory mechanism of protein function by S-oxidation/thiolation, we selected R-enolase as an example because (i) among glycolitic enzymes, R-enolase is generally detected as a target of oxidative modification (21-24) and (ii) oxidative modification of this enzyme has been implicated in Alzheimer’s disease (22). The incorporation of biotin-cysteine into endogenous R-enolase was confirmed by pull-down with NeutrAvidin beads followed by Western blot with an anti-R-enolase antibody. To this end, the SH-SY5Y cells were treated with 15d-PGJ2 (10 µM) for 1 h followed by treatment with biotin-cysteine (100 µM), and the cell lysate was incubated with NeutrAvidin beads. After they were washed with lysis buffer, the proteins bound to the resin through biotin-cysteine were eluted with SDS-PAGE sample buffer, and R-enolase was detected by immunoblot analysis with the anti-R-enolase antibody (Figure 4A). Alternatively, cell lysates were subjected to immunoprecipitation with an anti-R-enolase antibody, and the presence of protein-bound biotin-cysteine was detected by Western blot analysis with HRP-conjugated NeutrAvidin (Figure 4B). Taken together, endogenous R-enolase was confirmed to be oxidatively modified to an appreciable extent during the 15d-PGJ2-stimulated oxidative stress. Reversible Inactivation of S-Oxidized r-Enolase by Mixed Disulfide Formation with GSH. In the presence of various reactive species, GSH can form disulfide links with protein cysteine residues, a posttranslational modification referred to as S-glutathiolation (25-27). The reversible modification of cysteines by GSH-disulfide linkage is thought to serve a protective function, masking critical protein sulfhydryls from more

Figure 4. Cysteine-targeted oxidation of endogenous R-enolase in SH-SY5Y cells exposed to 15d-PGJ2. SH-SY5Y cells were incubated with 10 µM 15d-PGJ2 for 1 h and then treated with 100 µM biotin-cysteine for 15 min. Cell lysates were incubated with immobilized NeutrAvidin or with anti-R-enolase-sepharose, as indicated. The presence of S-thiolated R-enolase was detected by immunoblot analysis (A), and the incorporation of biotincysteine into R-enolase immunoprecipitates was detected with HRP-NeutrAvidin and ECL (B). Abbreviation: NSE, neuron specific enolase.

extensive or irreversible oxidation. To this purpose, we investigated the effect of S-glutathiolation on the protein function of a glycolytic enzyme R-enolase. Purified R-enolase was treated with the thiol oxidant diamide (1 mM) for 15 min at 25 °C in the absence or presence of GSH (1 mM), and the amount of enolase activity remaining after treatment was determined. As shown in Figure 5, the addition of GSH to incubations with diamide substantially enhances the inhibition of R-enolase activity. For example, GSH increased the inhibition of R-enolase

1318

Chem. Res. Toxicol., Vol. 17, No. 10, 2004

Ishii and Uchida

Figure 5. Regulation of R-enolase by S-oxidation/thiolation. R-Enolase (0.3 mg) was incubated with diamide, GSH, or diamide plus GSH in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. Attempts to reverse the effect of diamide plus GSH on R-enolase were carried out by adding DTT to filtered enzyme samples after treatment with diamide plus GSH, and incubation was continued for an additional 30 min at 25 °C before assay. Concentrations of the reagents used are as follows: diamide, 1 mM; GSH, 1 mM; and DTT, 10 mM.

activity to 60.8% of control in the presence of 1 mM diamide, whereas without GSH, diamide reduced the enolase activity to only 89.1% of the control. The loss in activity most probably occurred due to S-thiolation of R-enolase, since the R-enolase activity was not affected in the presence of GSH (1 mM). This proposition was confirmed by the observation that when the sulfhydrylreducing agent DTT (1 mM, 30 min) was added to incubations prior to diamide/GSH, the inhibition of R-enolase was completely prevented (Figure 5). Glutathiolation of S-Oxidized r-Enolase. To investigate the mechanism by which GSH enhances R-enolase inhibition under sulfhydryl oxidizing conditions, the treatment of the enzyme with GSH/diamide was modified to include biotin-GSH, and the binding of biotin-GSH to the oxidized R-enolase was characterized. As shown in Figure 6A, oxidative modification of R-enolase upon treatment with diamide was indicated by the nonreducing SDS-PAGE. This modification was suggested to be due to sulfhydryl oxidation, because the protein bands, corresponding to the oxidized R-enolase, disappeared and only a 47 kDa protein, corresponding to intact R-enolase, was detected under the reducing condition (panel B). Western blot analysis also showed that under nonreducing (but denaturing) conditions, R-enolase electrophoresed as the 47 kDa monomer was significantly modified with biotin-GSH/diamide (panel C). If DTT was added to reactions before biotin-GSH/diamide, the enzyme was not modified by the incorporation of the probe (panel D). Similarly, if either DTT was added to enzyme preparations after biotin-GSH/diamide treatment, the biotinGSH-mediated labeling of R-enolase was reversed (data not shown). To determine the increase in the molecular mass of R-enolase due to glutathiolation, R-enolase incubated with GSH/diamide was directly analyzed by MALDI-TOF MS. As shown in Figure 6B, the MALDI-TOF MS analysis of the native R-enolase revealed a peak of m/z 47669.8. When R-enolase was incubated with diamide/ GSH in 50 mM sodium phosphate buffer (pH 7.2) for 15

Figure 6. Reversible binding of biotin-GSH to S-oxidized R-enolase. R-Enolase (0.3 mg) was incubated with diamide, biotin-GSH, or diamide plus biotin-GSH in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. Concentrations of the reagents used are as follows: diamide, 1 mM; GSH, 1 mM; and DTT, 10 mM. (A) After the treatment of R-enolase samples with diamide, biotin-GSH, or diamide plus biotin-GSH as described, an aliquot of the sample was added to the sample buffer with or without a reducing agent (DTT) for 5 min at 100 °C. Samples (3 µg/well) were then separated by 10% SDS-PAGE. Gels were then stained with SYPRO Red protein gel stain and transblotted onto a nitrocellulose membrane. Immunoblot analyses were carried out as described in the Experimental Procedures. (B) MALDI-TOF MS analysis of native (left) and S-thiolated (right) R-enolase.

min at 25 °C, the peak (m/z 48021.6) corresponding to the addition of approximately one molecule of GSH per protein was observed. Treatment of diamide/GSH-treated R-enolase with DTT prior to MALDI-TOF MS analysis removed the GSH (data not shown). Identification of the S-Oxidation/Thiolation Sites in r-Enolase. To confirm the S-oxidation/thiolation sites in R-enolase, the native and diamide/GSH-treated enolase were digested with trypsin and then analyzed by MALDITOF MS. Peptide mass mapping by MALDI-TOF MS analysis of the tryptic fragments from the native R-enolase provided identification of the four fragments, including one or two cysteine residues. Relative to the calculated mass of the unmodified peptide, two peptides (P-1 and P-2), which showed an increased mass of +305 Da corresponding to the addition of a single molecule of GSH, were detected by MALDI-TOF MS analysis. P-1 gave a peak of m/z 1768.9, representing the glutathiolated peptide fragment (FGANAILGVSLAVCK; sequence

Targets of Oxidative Stress Induced by 15d-PGJ2

Chem. Res. Toxicol., Vol. 17, No. 10, 2004 1319

Figure 8. MALDI-TOF MS analysis of the affinity-purified biotin-GSH-labeled peptides. R-Enolase (0.3 mg) was incubated with diamide, biotin-GSH, or diamide plus biotin-GSH in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. Both native (lower chromatogram) and diamide/biotin-GSH-treated (upper chromatogram) R-enolases were digested with trypsin, and then, the biotin-GSH-labeled peptides were affinity-purified using StreptAvidin-Plus beads. After biotin-GSH-tagged peptides bound to the beads were treated with DTT, free peptide thiols were alkylated with iodacetamide to avoid uncontrolled disulfide formation and analyzed by MALDI-TOF peptide mass mapping.

Figure 7. MALDI-TOF MS analysis of the tryptic fragments from S-oxidized/thiolated R-enolase. R-Enolase (0.3 mg) was incubated with diamide, biotin-GSH, or diamide plus biotinGSH in 50 mM phosphate buffer (pH 7.2) for 15 min at 25 °C. After the treatment of R-enolase samples with diamide, biotinGSH, or diamide plus biotin-GSH as described, an aliquot of the sample was added to the sample buffer with or without a reducing agent (DTT) for 5 min at 100 °C. Concentrations of the reagents used are as follows: diamide, 1 mM; GSH, 1 mM; and DTT, 10 mM. (A) Detection of a S-thiolated peptide (P-1), which gave a peak of m/z 1768.9 as the glutathiolated peptide fragment. (B) Detection of a S-thiolated peptide (P-2), which gave a peak of m/z 2603.3 as the glutathiolated peptide fragment.

105-119) (Figure 7A), whereas P-2 gave a peak of m/z 2603.3, representing the glutathiolated peptide fragment (SGETEDTFIADLVVGLCTGQIK; sequence 372-393) (Figure 7B). Treatment of diamide/GSH-treated R-enolase with DTT prior to MALDI-TOF MS analysis resulted in the disappearance of P-1 and P-2 (data not shown). These data indicate that the S-oxidation sites in R-enolase represent Cys-118 and Cys-388. To further confirm that Cys-118 and Cys-388 are oxidized, the S-oxidized R-enolase treated with biotinGSH was subjected to trypsin digestion, and the molecular masses of the tryptic peptides were determined by

MALDI-TOF MS analysis after affinity purification of biotin-GSH-labeled peptides followed by their reduction and alkylation. Both native and diamide/biotin-GSHtreated R-enolases were digested with trypsin, and then, the biotin-GSH-labeled peptides were affinity-purified using StreptAvidin-Plus beads. After biotin-GSH-tagged peptides bound to the beads were treated with DTT, free peptide thiols were alkylated with iodacetamide to avoid uncontrolled disulfide formation and analyzed by MALDITOF peptide mass mapping. As shown in Figure 8, MALDI-TOF MS analysis of the purified peptides from the diamide/GSH-treated R-enolase detected two fragments (P-3 and P-4), which gave a peak of m/z 1519.6 and 2353.3, respectively. These peaks represented the addition of a 57 Da adduct, in accord with acetamide derivatization of the thiol groups of FGANAILGVSLAVCK (P-3) and SGETEDTFIADLVVGLCTGQIK (P-4). These data indicate that the peptide sequences of P-3 and P-4 were identical to those of P-1 and P-2, respectively. Thus, Cys-118 and Cys-388 in R-enolase are the sole targets of S-oxidation, and the remaining cysteine-containing peptides at Cys-336, Cys-338, Cys-356, and Cys-398 were not modified by diamide/GSH.

Discussion In the present study, to investigate the correlation between the redox regulation and 15d-PGJ2-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins. To this end, we utilized biotin-cysteine as the probe in the investigation of cysteine-targeted oxidation of proteins. The presence of a biotin tag on proteins, which become thiolated, allowed a range of investigative procedures to be carried out, which exploit the high affinity of biotin for avidin derivatives. Thus, the S-oxidized proteins could be detected on nonreducing Western blots using streptavidinHRP, and these were quantified via digitization. They

1320

Chem. Res. Toxicol., Vol. 17, No. 10, 2004

could also be purified using a streptavidin affinity matrix and then identified using Western immunoblotting. Here, we showed that cysteine-targeted protein oxidation induced by exposure to 15d-PGJ2 oxidatively modified a range of proteins involved in various processes, including cytoskeletal proteins, glycolytic enzymes, redox enzymes, and chaperone protein (Figure 2 and Table 1). The observation that oxidation targets particular enzymes indicates that this protein modification may serve a regulatory role, rather than a simple function in the protection of protein SH groups against irreversible oxidation. Proteomics identification of S-oxidized proteins in SHSY5Y cells exposed to 15d-PGJ2 revealed that a glycolytic enzyme R-enolase was one of the targets of S-oxidation. In addition, S-oxidation of endogenous R-enolase during the 15d-PGJ2-stimulated intracellular oxidative stress was also confirmed (Figure 4). Enolase catalyzes the interconversion of 2-phosphoglycerate to phosphoenolpyruvate. Higher vertebrates possess three enolase genes, encoding R-, β-, and γ-subunits (28). Nonneuronal enolase, a homodimer of 47 kDa subunits, is the major isoform during embryogenesis and persists in nearly every adult tissue (29, 30). The γ-subunit, also 47 kDa, is found in the γγ and γR dimers known collectively as neuron specific enolase (31, 32). Neuron specific enolase is expressed mainly in normal and neoplastic cells of neuronal and neuroendocrine origin (33, 34) but has been found in diverse mammalian cell types (35, 36). It has been reported that R-enolase is the target of glutathiolation in human T-cell blasts exposed to oxidative stress (diamide or hydrogen peroxide) (21). This and the observation that the synthesis of glycolytic enzymes, including R-enolase, is repressed in response to oxidative stress (37) suggest that cells respond to oxidative stress by inhibiting glycolytic enzymes via S-thiolation and switch off gene expression to prevent the synthesis of any new enzymes. Combining regulation at the levels of protein modification and gene expression would provide a rapid means of reversibly inhibiting the flux through glycolysis. Regulation of glycolysis by protein S-thiolation may be beneficial during conditions of oxidative stress since it would result in an increased flux of glucose equivalents through the pentose phosphate pathway leading to the generation of NADPH (38, 39). To investigate the molecular mechanism by which S-oxidation/thiolation enhances R-enolase inhibition under sulfhydryl oxidizing conditions, we characterized the binding of GSH to the oxidized R-enolase in vitro. R-Enolase was slightly inhibited by the specific thiol oxidant diamide and this inhibition of enzyme activity was significantly enhanced by GSH (Figure 5). The characteristics of the inhibition of R-enolase by diamide in the presence of GSH were entirely consistent with other studies that have established that proteins can be modified by disulfide linkages with low molecular weight thiols, such as GSH (25-27). To confirm the oxidation sensitive cysteines in R-enolase, native and diamide/GSHtreated enolase were digested with trypsin and then analyzed by MALDI-TOF MS. MALDI-TOF MS detected a mass shift indicative of deglutathiolation of the peptide fragment and allowing unequivocal assignment of Cys118 and Cys-388 as sites of oxidation (Figures 7 and 8). R-Enolase has five cysteine residues per 47 kDa monomer. The MALDI-TOF MS analysis showed the site of the modification to be exclusively at Cys-118 and Cys-

Ishii and Uchida

388. These data suggest that R-enolase may not be randomly oxidized but instead may undergo oxidative modification in specific environments. S-Thiolation is an oxidative, reversible posttranslational modification of cysteine residues of proteins and can be viewed as a protective mechanism that guards against the terminal or irreversible oxidation of these residues. Protein S-thiolation can be directly coupled to cellular redox status and has no absolute requirement for specialized regulatory enzymes, although thiol transferase enzymes can catalyze these reactions (40). It has long been recognized that low molecular weight thiols, such as GSH, can interact in a reversible manner with the cysteine sulfhydryl groups in many cellular proteins (25, 41). In particular, protein S-thiolation/dethiolation is a dynamic process that occurs in cells under physiological conditions as well as following exposure to an oxidative stress (26, 27, 42). Models have been proposed in which the modification of proteins by S-thiolation does not require an enzymatic activity but proceeds via the reaction of partially oxidized protein sulfydryls (thiyl radical or sulfenic acid intermediates) with thiols such as cysteine or GSH or by thiol/disulfide exchange reactions with the oxidized disulfide form of glutathione (GSSG) (27). A variety of proteins that become Sthiolated in response to cellular stress have been detected in mammalian cells. These include key metabolic enzymes such as carbonic anhydrase, glycogen phosphorylase, creatine kinase, glyceraldehyde 3-phosphate dehydrogenase, GSH transferase, and superoxide dismutase as well as structural and transport proteins such as hemoglobin, actin, and crystallin (reviewed in ref 4). Studies have also suggested that this modification may be involved in the regulation of the function and activity of proteins, including the human immunodeficiency virus type 1 protease (43), ubiquitin-conjugating enzymes in bovine retina cells (44), and DNA binding by the transcription factor c-Jun (45). The proteins that were S-thiolated are those with reactive cysteines, which may be particularly susceptible to a range of oxidative modification, not just S-thiolation. The actual mode of oxidation may involve modification by a range of species, including GSH, cysteine, homocysteine, nitrosoglutathione, GSH sulfonamide, GSH disulfide S-oxide, nitric oxide, hypochlorous acid, or other oxidizing species. Indeed, comparisons of the patterns of S-thiolated and carbonylated proteins in SH-SY5Y cells exposed to 15d-PGJ2 reveal that both modifications seem to have common protein substrates (Ishii, T., and Uchida, K. Unpublished observations). It has also been shown that the cysteines within the G-protein Ha-Ras can be both S-thiolated and S-nitrosylated (46). The type of oxidative cysteine modification, which takes place during an oxidative insult, may depend on many factors, including the nature, complexity, and intensity of the prevailing oxidant stress. In summary, we analyzed S-oxidized proteins generated in response to intracellular oxidative stress stimulated with an endogenous electrophile and identified 27 proteins, including cytoskeletal proteins, glycolytic enzymes, redox enzymes, and chaperone proteins, as substrates for reversible cysteine-targeted protein oxidation. In addition, to investigate the regulatory mechanism of the protein function by S-oxidation/thiolation, we characterized the binding of GSH to the oxidized R-enolase and identified two cysteine residues, Cys-118 and Cys-

Targets of Oxidative Stress Induced by 15d-PGJ2

388, as the oxidation sensitive sites, which may play a role in the regulation of the biological activities of the protein and may be regulated by reversible S-oxidation/ thiolation reaction. The results of this study suggest that cysteine-targeted oxidation/thiolation plays a critical role in the regulation of protein function under conditions of electrophile-induced oxidative stress.

Chem. Res. Toxicol., Vol. 17, No. 10, 2004 1321

(17)

(18)

Acknowledgment. This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology, and by the COE Program in the 21st Century in Japan.

(19)

Supporting Information Available: Peptide mass fingerprinting analysis of an S-oxidized/thiolated protein. This material is available free of charge via the Internet at http:// pubs.acs.org.

(21)

References

(22)

(1) Halliwell, B., and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine, 2nd ed., Clarendon Press, Oxford. (2) Sundaresan, M., Yu, Z.-X., Ferrans, V. J., Irani, K., and Finkel, T. (1995) Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296-299. (3) Giles, H., and Leff, P. (1988) The biology and pharmacology of D2. Prostaglandins 35, 277-300. (4) Fitzpatrick, F. A., and Wynalda, M. A. (1983) Albumin-catalyzed metabolism of prostaglandin D2. Identification of products formed in vitro. J. Biol. Chem. 258, 11713-11718. (5) Kikawa, Y., Narumiya, S., Fukushima, M., Wakatsuka, H., and Hayaishi, O. (1984) 9-Deoxy-∆9,∆12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma. Proc. Natl. Acad. U.S.A. 81, 1317-1321. (6) Hirata, Y., Hayashi, H., Ito, S., Kikawa, Y., Ishibashi, M., Sudo, M., Miyazaki, H., Fukushima, M., Narumiya, S., and Hayashi, O. (1988) Occurrence of 9-deoxy-∆9,∆12-13,14-dihydroprostaglandin D2 in human urine. J. Biol. Chem. 263, 16619-16625. (7) Shibata, T., Kondo, M., Osawa, T., Shibata, N., Kobayashi, M., and Uchida, K. (2002) 15-Deoxy-∆12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J. Biol. Chem. 277, 10459-10466. (8) Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K. (1998) The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation. Nature 391, 7982. (9) Jiang, C., Ting, A. T., and Seed, B. (1998) PPAR-γ agonists inhibit production of monocyte inflammatory cytokines. Nature 391, 8286. (10) Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., and Santoro, M. G. (2000) Antiinflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase. Nature 403, 103-108. (11) Straus, D., Pascual, G., Li, M., Welch, J. S., Ricote, M., Hsiang, C.-H., Sengchanthalangsy, L. L., Ghosh, G., and Glass, C. K. (2000) 15-Deoxy-∆12,14-prostaglandin J2 inhibits multiple steps in the NF-κB signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 97, 4844-4849. (12) Bishop-Bailey, D., and Hla, T. (1999) Endothelial cell apoptosis induced by the peroxisome proliferator-activated receptor (PPAR) ligand 15-deoxy-∆12,14-prostaglandin J2. J. Biol. Chem. 274, 17042-17048. (13) Kawamoto, Y., Nakamura, Y., Naito, Y., Torii, Y., Kumagai, T., Osawa, T., Ohigashi, H., Satoh, K., Imagawa, M., and Uchida, K. (2000) Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzyme: 15-Deoxy-D12,14-prostaglandin J2-induced expression of glutathione S-transferases. J. Biol. Chem. 275, 11291-11299. (14) Kondo, M., Shibata, T., Kumagai, T., Osawa, T., Shibata, N., Kobayashi, M., Sasaki, S., Iwata, M., Noguchi, N., and Uchida, K. (2002) 15-Deoxy-∆12,14-prostaglandin J2: The endogenous electrophile that induces neuronal apoptosis. Proc. Natl. Acad. Sci. U.S.A. 99, 7367-7372. (15) Cernuda-Morollon, E., Pineda-Molina, E., Canada, F. J., and Perez-Sala, D. (2001) 15-Deoxy-∆12,14-prostaglandin J2 inhibition of NF-κB-DNA binding through covalent modification of the p50 subunit. J. Biol. Chem. 276, 35530-35536. (16) Kondo, M., Shibata, T., Kumagai, T., Osawa, T., Shibata, N., Kobayashi, M., Sasaki, S., Iwata, M., Noguchi, N., and Uchida,

(20)

(23)

(24)

(25)

(26) (27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

K. (2002) 15-Deoxy-∆12,14-prostaglandin J2: The endogenous electrophile that induces neuronal apoptosis. Proc. Natl. Acad. Sci. U.S.A. 99, 7367-7372. Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J. Biol. Chem. 276, 12076-12083. Eaton, P., Byers, H. L., Leeds, N., Ward, M. A., and Shattock, M. J. (2002) Detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. J. Biol. Chem. 277, 9806-9811. Humphries, K. M., Juliano, C., and Taylor S. S. (2002) Regulation of cAMP-dependent protein kinase activity by glutathionylation. J. Biol. Chem. 277, 43505-43511. Pancholi, H., and Fischetti, V. A. (1998) R-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. Biol. Chem. 273, 14503-14515. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 99, 3505-3510. Castegna, A., Aksenov, M., Thongboonkerd, V., Klein, J. B., Pierce, W. M., Booze, R., Markesbery, W. R., and Butterfield, D. A. (2002) Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part II: Dihydropyrimidinase-related protein 2, R-enolase and heat shock cognate 71. J. Neurochem. 82, 1524-3152. Cabiscol, E., Piulats, E., Echave, P., Herrero, E., and Ros, J. (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J. Biol. Chem. 275, 27393-27398. Tamarit, J., Cabiscol, E., and Ros, J. (1998) Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress. J. Biol. Chem. 273, 3027-3032. Klatt, P., and Lamas, S. (2000) Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur. J. Biochem. 267, 4928-4944. Thomas, J. A., Chai, Y. C., and Jung, C. H. (1994) Protein S-thiolation and dethiolation. Methods Enzymol. 233, 385-395. Thomas, J. A., Poland, B., and Honzatko, R. (1995) Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. Arch. Biochem. Biophys. 319, 1-9. Peshavaria, M., Quinn, G. B., Reeves, I., Hinks, L. J., and Day, I. M. (1990) Molecular biology of the human enolase gene family: Nerve (γ), muscle (β) and general (R) isoforms. Biochem. Soc. Trans. 18, 254-255. Katagiri, T., Feng, X., Ichikawa, T., Usui, H., Takahashi, Y., and Kumanishi, T. (1993) Neuron-specific enolase (NSE) and nonneuronal enolase (NNE) mRNAs are coexpressed in neurons of the rat cerebellum: in situ hybridization histochemistry. Brain Res. Mol. Brain Res. 19, 1-8. Marangos, P. J., Schmechel, D. E., Parma, A. M., and Goodwin, F. K. (1980) Developmental profile of neuron-specific (NSE) and nonneuronal (NNE) enolase. Brain Res. 190, 185-193. Tanaka, M., Sugisaki, K., and Nakashima, K. (1985) Switching in levels of translatable mRNAs for enolase isozymes during development of chicken skeletal muscle. Biochem. Biophys. Res. Commun. 133, 868-872. Keller, A., Berod, A., Dussaillant, M., Lamande, N., Gros, F., and Lucas, M. (1994) Coexpression of R and γ enolase genes in neurons of adult rat brain. J. Neurosci. Res. 38, 493-504. Gerbitz, K. D., Summer, J., Schumacher, I., Arnold, H., Kraft, A., and Mross, K. (1986) Enolase isoenzymes as tumour markers. J. Clin. Chem. Clin. Biochem. 24, 1009-1016. Isawe, K., Nagasaka, A., Kato, K., Ohtani, S., Nagatsu, I., Ohyayma, T., Nakai, A., Aono, T., Nakagawa, H., and Shinoda, S. (1986) Enolase subunits in patients with neuroendocrine tumors. J. Clin. Endocrinol. Metab. 63, 94-101. Schmechel, D. E. (1985) γ-Subunit of the glycolytic enzyme enolase: Nonspecific or neuron specific? Lab. Invest. 52, 239242. Haimoto, H., Takahashi, Y., Koshikawa, T., Nagura, H., and Kato, K. (1985) Immunohistochemical localization of gamma-enolase in normal human tissues other than nervous and neuroendocrine tissues. Lab. Invest. 52, 257-263. Shenton, D., and Grant, C. M. (2003) Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae. Biochem. J. 374, 513-519. Schuppe-Koistinen, I., Moldeus, P., Bergman, T., and Cotgreave, I. A. (1994) S-Thiolation of human endothelial cell glyceraldehyde3-phosphate dehydrogenase after hydrogen peroxide treatment. Eur. J. Biochem. 221, 1033-1037.

1322

Chem. Res. Toxicol., Vol. 17, No. 10, 2004

(39) Ravichandran, V., Seres, T., Moriguchi, T., Thomas, J. A., and Johnston, R. B. (1994) S-Thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes. J. Biol. Chem. 269, 25010-25015. (40) Grant, C. M. (2001) Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 39, 533-541. (41) Cotgreave, I. A., and Gerdes, R. G. (1998) Recent trends in glutathione biochemistry: Glutathione-protein interactions: A molecular link between oxidative stress and cell proliferation? Biochem. Biophys. Res. Commun. 242, 1-9. (42) Seres, T., Ravichandran, V., Moriguchi, T., Rokutan, K., Thomas, J. A., and Johnston, R. B. (1996) Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible posttranslational modification with potential for buffering the effects of oxidant stress. J. Immunol. 156, 19731980.

Ishii and Uchida (43) Davis, D. A., Newcomb, F. M., Starke, D. W., Ott, D. E., Mieyal, J. J., and Yarchoan, R. (1997) Thioltransferase (glutaredoxin) is detected within HIV-1 and can regulate the activity of glutathionylated HIV-1 protease in vitro. J. Biol. Chem. 272, 25935-25940. (44) Obin, M., Shang, F., Gong, X., Handelman, G., Blumberg, J., and Taylor, A. (1998) Redox regulation of ubiquitin-conjugating enzymes: Mechanistic insights using the thiol-specific oxidant diamide. FASEB J. 12, 561-569. (45) Klatt, P., Molina, E. P., De, Lacoba, M. G., Padilla, C. A., Martinez-Galisteo, E., Barcena, J. A., and Lamas, S. (1999) Redox regulation of c-Jun DNA binding by reversible S-glutathiolation. FASEB J. 13, 1481-1490. (46) Mallis, R. J., Buss, J. E., and Thomas, J. A. (2001) Oxidative modification of H-ras: S-Thiolation and S-nitrosylation of reactive cysteines. Biochem. J. 355, 145-153.

TX049860+