Ebselen, a Seleno-organic Antioxidant, as an Electrophile - American

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Chem. Res. Toxicol. 2006, 19, 1196-1204

Ebselen, a Seleno-organic Antioxidant, as an Electrophile Toyo Sakurai,† Masaya Kanayama,† Takahiro Shibata,† Ken Itoh,‡ Akira Kobayashi,‡ Masayuki Yamamoto,‡ and Koji Uchida*,† Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan, and Center for Tsukuba AdVanced Research Alliance and Institute of Basic Medical Sciences, UniVersity of Tsukuba, Tsukuba 305-8577, Japan ReceiVed May 24, 2006

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the electrophilic potential of this antioxidant and established the mechanism of the cysteine-targeted oxidation of protein. In addition, using ebselen as an electrophilic probe, we characterized the cysteine residues required for posttranslational modification into an electrophile sensor protein in the phase 2 detoxification response. Ebselen showed a potent antioxidant effect against the spontaneous and 4-hydroxy2-nonenal-stimulated production of intracellular reactive oxygen species in rat liver epithelial RL34 cells. Meanwhile, upon in vitro incubation with a redox-active sulfhydryl protein (thioredoxin), ebselen showed a strong electrophilic potential of mediating the formation of selenenylsulfide and intra- and intermolecular disulfide linkages within the protein. By taking advantage of this antioxidant and electrophilic property of ebselen, we characterized posttranslational modification of Kelch-like ECH-associated protein 1 (Keap1), an electrophile sensor protein, which represses the ability of the transcription factor NF-E2-related factor 2 (Nrf2) upon induction of the phase 2 detoxification response. Ebselen potently induced the gene expression of a series of phase 2 enzymes in rat liver epithelial RL34 cells, which was associated with the formation of a high molecular weight complex of Keap1. Furthermore, a cysteine residue in Keap1, C151, was found to be uniquely required not only for the formation of the complex but also for the induction of the phase 2 response by ebselen. Thus, this unique antioxidant and electrophilic property of ebselen giving rise to the cysteine-targeted oxidation enabled us to evaluate the role of sensor cysteines in redox regulation of protein function under electrophile stress. Introduction Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one; see Figure 1] is a seleno-organic drug that has been extensively studied during the past decade. A substantial part of the pharmacological profile of ebselen appears to be due to its action as an antioxidant. It has been well-documented that ebselen exhibits a weak glutathione peroxidase (GPx)1-like activity in vitro (13). More recently, Holmgren and his colleagues have shown that ebselen is an excellent substrate for mammalian thioredoxin reductase (TrxR) and a highly efficient oxidant of reduced thioredoxin (Trx), and it catalyzed the H2O2 reduction (4). On the other hand, ebselen also displays a unique pattern of chemical reactions, which is not restricted to the GPx reaction (for review, see ref 5). Indeed, it inhibits at low concentrations a number of enzymes involved in inflammation, such as lipoxygenases, nitricoxide synthases, NADPH oxidase, protein kinase C, and H+/K+-ATPase (6). Recent animal studies showed the antioxidant and anti-inflammatory actions of ebselen in a * To whom correspondence should be addressed. E-mail: [email protected]. † Nagoya University. ‡ University of Tsukuba. 1 Abbreviations: ARE, antioxidant response element; DTT, dithiothreitol; EpRE, electrophile response element; GPx, glutathione peroxidase; GSH, glutathione, reduced form; GSSG, glutathione, oxidized form; GST, glutathione S-transferases; HNE, 4-hydroxy-2-nonenal; HRP, horseradish peroxidase; Keap1, Kelch-like ECH-associated protein 1; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; Nrf2, NF-E2related factor 2; PBS, phosphate buffered saline; ROS, reactive oxygen species; Trx, thioredoxin; TrxR, thioredoxin reductase.

Figure 1. Chemical structure of ebselen.

variety of experimental animal models (7-9). In addition, ebselen has been used in the treatment of patients with acute ischemic stroke (10, 11) or delayed neurological deficits after aneurysmal subarachnoid hemorrhage (12). The results of these clinical trials indicate the benefit of ebselen as a neuroprotective agent. Xenobiotic metabolizing enzymes play a major role in regulating the toxic, oxidative damaging, mutagenic, and neoplastic effects of chemical carcinogens. Mounting evidence has indicated that the induction of phase 2 detoxification enzymes, such as glutathione S-transferases (GSTs), results in protection against toxicity and chemical carcinogenesis, especially during the initiation phase. The induction of phase 2 enzymes is reported to be evoked by an extraordinary variety of chemical agents, including Michael reaction acceptors, diphenols, quinones, isothiocyanates, peroxides, vicinal dimercaptans, and others (13-15). With few exceptions, these agents are electrophiles, and accordingly, many of these inducers are substrates for the phase 2 detoxification enzymes. The transcriptional activation of the phase 2 enzymes has been traced to a cis-acting transcriptional enhancer called the antioxidant response element (ARE) (16), or alternatively, the electrophile response element (EpRE) (17). NF-E2-related factor 2 (Nrf2),

10.1021/tx0601105 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/12/2006

Ebselen as an Electrophile

a member of the NF-E2 family of nuclear basic leucine zipper transcription factors, binds to the ARE and accelerates the transcription of the cognate genes (18-20). Under basal conditions, Kelch-like ECH-associated protein 1 (Keap1), a protein associated with the actin cytoskeleton, binds very tightly to Nrf2, anchors this transcription factor in the cytoplasm, and targets it for ubiquitination and proteasome degradation, thereby repressing the ability of Nrf2 to induce phase 2 detoxification enzyme genes (21-26). Because of the high content of the cysteine residues, Keap1 has been suggested to be a direct sensor for phase 2 inducers. Among several candidate cysteines for the actual targets for inducers, C273 and C288 have appeared critical not only for the inducer sensing function of Keap1 but also for its repressor activity (27). Moreover, C151 has also been identified as a target site for modification by certain inducers (28, 29). In the present study, we shed light on the electrophilic function of ebselen and unexpectedly found that, despite the down-regulation of intracellular oxidative stress, ebselen potently induced the cysteine-targeted oxidation of cellular proteins. To gain a better understanding of the molecular basis of the ROSindependent electrophile stress, the mechanism for the ebseleninduced S-oxidation was characterized using a representative redox-active protein, Trx. Moreover, taking advantage of this unique antioxidant and electrophilic property of ebselen, we characterized cysteine residues required for the posttranslational modification to Keap1 in the phase 2 detoxification response.

Materials and Methods Materials. Ebselen was purchased from Sigma. Biotinylated cysteine (biotin-cysteine) was prepared as previously described (30, 31). Horseradish peroxidase (HRP)-conjugated NeutrAvidin and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Biosciences. 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. Human recombinant Trx was kindly provided by Dr. Junji Yodoi of Kyoto University. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was from Molecular Probes, Inc. (Eugene, OR). The stock solutions of 4-hydroxy-2-nonenal (HNE) were prepared by the acid treatment (1 mM HCl) of HNE dimethylacetal, which was synthesized according to the procedure of De Montarby et al. (32). Anti-Nrf2 polyclonal antibodies were obtained from Santacruz (Santa Cruz, CA). Plasmid Construction. For the reporter transfection assay, we used a promoterless luciferase plasmid vector (pGVB; Nippon Gene, Toyama, Japan) that had the minimal promoter sequence of the GST A1 (33), quinone reductase (34), or hemeoxygenase-1 (35) gene, containing a GC box and a TATA box. Full-length mouse Keap1 cDNA was subcloned into pcDNA3 (Invitrogen) vector. The mutant Keap1 genes containing alanine and serine codons substituted for specific cysteine codons were constructed. In the current experiments, we focused our attention on one cysteine residue (C151) within the BTB domain and two cysteine residues (C273 and C288) within the intervening region of Keap1. They were selected on the basis of a recent report that these cysteine residues preferentially react with thiol-specific chemicals in vitro (29, 36). All constructs were confirmed by sequencing. Transfection Experiments and Luciferase Assay. The RL34 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and seeded in 4 × 104/well in six well dishes 2-18 h before transfection. The cells were transfected with plasmids using Lipofectamine2000 (Invitrogen) according to the manufacturer‘s instructions. Twentyfour hours after the transfection, the cells were treated with ebselen (10 µM). After 24 h, the Luciferase assay was performed by

Chem. Res. Toxicol., Vol. 19, No. 9, 2006 1197 utilizing the Dual Luciferase Assay System (Nippon Gene) following the supplier’s protocol and measured in a Mini Lumar LB9560 (Berthold, Bad Wildbad, Germany). Transfection efficiencies were routinely normalized by the activity of a cotransfected Renilla luciferase (5 µg). Normally, three independent experiments, each carried out in duplicate, were performed. Cell Culture. RL34 cells were obtained from the Japanese Cancer Research Resources Bank. The cells were grown as monolayer cultures in DMEM supplemented with 5% heatinactivated FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (588 µg/mL), and 0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2. Flow Cytometry Analysis of Intracellular ROS Production. DCFH-DA was used to measure the ROS (37, 38). Cells were incubated with 10 µM 2′,7′-dichlorofluorescein diacetate (dissolved in DMSO) for 30 min at 37 °C and then treated with different agents for an additional 1 h at 37 °C. After they were chilled on ice, the cells were washed with ice-cold phosphate-buffered saline (PBS), scraped from the plate, and resuspended at 1 × 106 cells/mL in PBS containing 10 mM EDTA. The fluorescence was measured using a flow cytometer (Epics XL, Beckman Coulter). Ebselen Modification of Trx. Human recombinant Trx was incubated with 0.5 mM ebselen in sodium phosphate buffer (pH 7.2) for 30 min at 37 °C. The native and ebselen-modified Trx were incubated with SDS sample buffer with or without a reducing agent for 5 min at 100 °C. The samples ware separated by 10% SDS-polyacrylamide gel electrophoresis. The gel was transblotted onto a nitrocellulose or PVDF membrane, incubated with Blockace for blocking, washed, and incubated with the HRP-NeutrAvidin. This procedure was followed by the addition of ECL reagents. The bands were visualized by LumiVision Pro 400EX (AISIN SEIKI Co., Ltd., Aichi, Japan). For identification of the target cysteines in Trx, the native and ebselen-modified Trx (1 mg/mL) were digested with modified trypsin in 20 µL of 50 mM Tris-HCl buffer (pH 8.8) at 37 °C for 24 h using an enzyme:substrate ratio of 1:100 (w/w). The trypsin-digested Trx was mixed with 2.5 mg/mL R-cyano-4-hydroxycinnamic acid (Sigma) containing 50% acetonitrile and 0.1% trifluoroacetic acid and dried on stainless steel targets at room temperature and pressure. The analyses were performed using a 4700 Proteomics Analyzer matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF MS) spectrometer (Applied Biosystems) with a nitrogen laser (337 nm). All analyses were run in the positive ion mode, and the instrument was immediately calibrated prior to each series of studies. RT-PCR. The total RNA was isolated from the cells using TRIZOL reagent (Invitrogen Co., Carlsbad, CA) according to the manufacturer’s protocol and spectrophotometrically quantified. The total RNAs (5 µg) were reverse transcribed into cDNA and used for an RT-PCR analysis (QIAGEN, Hilden, Germany). GAPDH was used as an internal standard. The PCR products were separated in a 1% agarose gel, and the positive signals were quantified by densitometry analysis after staining with ethidium bromide. Two-Dimensional Gel Electrophoresis of S-Oxidized Proteins. To analyze the S-oxidized proteins, the RL34 cells were pretreated with 100 µM biotin-cysteine for 30 min and incubated with 20 µM ebselen for 1 h. The cells were then washed twice with PBS, lysed in lysis buffer (4% (w/v) CHAPS, 9 M urea, and 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. 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,

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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 the S-oxidized proteins was performed as previously described (39). Nonreducing-Reducing Two-Dimensional SDS-Polyacrylamide Gel Electrophoresis. Keap1 in RL34 cells treated with and without ebselen was analyzed by nonreducing-reducing twodimensional SDS-polyacrylamide gel electrophoresis (27). Keap1 was detected using the rabbit anti-Keap1 polyclonal antibody (Santacruz).

Results Antioxidant Action of Ebselen. We first examined the effect of ebselen (Figure 1) on the basal and stimulated productions of intracellular ROS in rat liver epithelial RL34 cells. The intracellular production of ROS was determined using the oxidation-sensitive fluorescent probe, DCFH-DA. In addition, electrophilic small chemicals, such as HNE, were used for the stimulated production of the intracellular ROS (Figure 2A). As shown in Figure 2B, a small, but significant, DCFH oxidation was observed even in the unstimulated (medium-treated) control cells, and this spontaneous production of ROS was significantly suppressed by treatment with ebselen. We then examined the antioxidant activity of ebselen in the electrophile (HNE)stimulated cells. Consistent with our previous findings (40), HNE at the concentrations of 25 and 50 µM stimulated the intracellular ROS production, whereas the HNE-stimulated productions of ROS were dramatically suppressed by the addition of ebselen (Figure 2C). On the basis of the fact that ebselen is an excellent substrate for mammalian Trx reductase and it catalyzes the H2O2 reduction, it is likely that the antioxidant action of ebselen may be, to a large extent, due to reactions with the Trx system (Figure 3) (4, 41). Electrophilic Action of Ebselen. On the other hand, despite the potential antioxidant effect against intracellular ROS production, ebselen reacts with glutathione (GSH) to form an ebselen selenenylsulfide, which in turn is slowly converted, in the presence of an excess of GSH, to ebselen selenol and the diselenide of ebselen (42, 43). In addition, it is also known that ebselen is a highly efficient oxidant of Trx (41). However, the mechanism for the binding of ebselen to protein thiols remains to be established. Hence, we characterized the ebselen-treated Trx by nonreducing and reducing SDS-polyacrylamide gel electrophoresis and by MALDI-TOF MS. When subjected to the nonreducing SDS-polyacrylamide gel electrophoresis, the native (unoxidized) form of Trx migrated as a single protein band of 12 kDa (Figure 4A). However, upon incubation with 0.5 mM ebselen in 50 mM sodium phosphate buffer (pH 7.2) at 37 °C, Trx was converted to a higher molecular weight protein species probably corresponding to the intermolecular crosslinking reactions. Meanwhile, the formation of these modified proteins was not detected during the reducing SDS-polyacrylamide gel electrophoresis (Figure 4B), suggesting that ebselen primarily formed selenenylsulfide (-Se-S-) and/or disulfide (-S-S-) linkages within Trx. To identify the ebselen-sensisitve cysteine residues in Trx, the ebselen-treated and untreated Trx were analyzed by MALDI-TOF MS. The native Trx was detected as a peak of m/z 1148, whereas the peak (m/z 1423.5) corresponding to the addition of one molecule of ebselen per protein was observed in the ebselen-treated Trx, suggesting that the ebselen modification site in the sequence is on C73 (Supporting Information, Figure S1). We also detected the peak (m/z 1148 × 2-2) corresponding to the intermolecular disulfide

Figure 2. Antioxidant action of ebselen. (A) Changes in intracellular ROS levels after exposure of RL34 cells to electrophiles. RL34 cells were incubated with DCFH-DA (10 µM) for 30 min and then treated with 10 µM tert-butylhydroquinone, HNE, or benzylisothiocyanate for 30 min. The fluorescence intensity was analyzed using a flow cytometer. (B) Effect of ebselen on spontaneous production of ROS in RL34 cells. The cells pretreated with ebselen (0-100 µM) for 1 h were incubated with 10 µM DCFH-DA for 30 min. Left panel, representative of three separate experiments. Right panel, averaged data of three separate experiments. (C) Effect of ebselen on HNE-induced production of ROS in RL34 cells. The cells pretreated with ebselen (10-100 µM) for 1 h were incubated with 10 µM DCFH-DA for 30 min and then exposed to 50 µM HNE for 30 min. Left panel, representative of three separate experiments. Right panel, averaged data of three separate experiments.

Figure 3. Antioxidant action of ebselen via the Trx system.

linkage at the same cysteine residue (C73) (Supporting Information, Figure S2). Moreover, we observed the peak (m/z 16242) corresponding to the intramolecular disulfide linkages


Figure 4. Oxidative modification of Trx by ebselen. Trx was incubated with 10 µM ebselen in sodium phosphate buffer (pH 7.2) at 37 °C. (A) Nonreducing SDS-polyacrylamide gel electrophoresis. (B) Reducing SDS-polyacrylamide gel electrophoresis. (C) A proposed mechanism for the cysteine-targeted oxidation by ebselen.

between C32 and C35 (Supporting Information, Figure S3), constituting the active center of Trx, and the peak (m/z 2719-2) corresponding to the intramolecular disulfide linkages between C62 and C69 (Supporting Information, Figure S4). These results suggest that the initially formed selenenylsulfide between ebselen and Trx was rapidly reduced by another highly reactive cysteine thiol of the protein to form a disulfide linkage (Figure 4C). Induction of ROS-Independent S-Oxidation of Cellular Proteins by Ebselen. To detect cellular proteins that undergo ROS-independent oxidation by ebselen, we utilized the biotincysteine as a molecular probe in the investigation of the cysteinetargeted protein S-oxidation (Figure 5A). This probe is thought to react not only with the intra- and intermolecular disulfide linkages but also with the selenenylsulfide linkage to form a biotin-cysteine-protein conjugate (Figure 5B). 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-thiolated proteins could be detected on nonreducing Western blots using streptavidin-HRP. To this end, RL34 cells treated with 100 µM biotin-cysteine for 30 min were incubated with 20 µM ebselen, and the biotin-cysteine-labeled (S-oxidized) proteins were separated on two-dimensional gel electrophoresis and analyzed by Western blot analysis probed with streptavidin-HRP. As shown in Figure 5C, several proteins were found to be oxidized in response to ebselen. The treatment of samples with 2-mercatoethanol almost completely abolished the signal (data not shown), indicating that the signals are detected as a consequence of a disulfide bond linking the biotin-cysteine to a protein. To identify the S-thiolated protein, spots were excised from the two-

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dimensional gels, subjected to trypsin digestion, and then successfully analyzed by a MALDI-TOF mass fingerprint analysis. Table 1 lists the identity of 16 protein spots. The identified proteins fall into several different functional classes, including cytoskeletal proteins (tublin, vimentin, and actin), glycolytic enzymes (enolase, malate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase), and chaperone proteins (Hsp27, Hsp47, HSP60, Hsp70, and peptidylproryl isomelase). Ebselen Activates the Keap1-Nrf2 Pathway. Electrophiles, having a chemical feature to react with the sulfhydryl groups by oxido-reduction, alkylation, or disulfide interchange, are inducers of the phase 2 enzymes (13). Like other electrophiles, ebselen significantly up-regulated a series of phase 2 enzymes (Figure 6A). In addition, we examined the involvement of ARE/ EpRE, a cis-acting element that is present in the promoters of the phase 2 enzyme genes (16, 17), in the ebselen-induced phase 2 enzyme gene expression, and found that ebselen potently stimulated the activity of the ARE/EpRE reporter genes (Figure 6B). These results are consistent with our previous findings that ebselen potentiates phase 2 enzyme activities in cultured hepatocytes and in mouse skin (9, 44). The Keap1-Nrf2 system is the major regulatory pathway of the phase 2 enzyme gene expression (21, 22, 45). Ebselen, indeed, induced nuclear localization of Nrf2 in the ebselen-treated RL34 cells (Figure 6C), suggesting that ebselen acts on this pathway. Ebselen-Induced Formation of a Keap1 Complex in RL34 Cells. Keap1, a repressor of the Nrf2 transactivation activity, is known to play a central role in regulating the adaptive responses to electrophilic and oxidative stresses. The high cysteine content of Keap1 suggested that it would be an excellent candidate as the sensor for inducers. It has been suggested that these cysteine residues constitute a multicomponent redoxsensitive switch that determines the ability of Keap1 to repress Nrf2-dependent transcription. Our observations that ebselen induced phase 2 enzyme gene expression and nuclear localization of Nrf2 suggest that ebselen may directly interact with the sensor protein Keap1, while we could not detect Keap1 as the target of ebselen in the proteomic analysis (Figure 5C and Table 1). Hence, taking advantage of the unique electrophilic property of ebselen giving rise to the formation of selenenylsulfide and disulfide linkages within proteins without generating ROS, we characterized the oxidative modification of Keap1 in ebselentreated cells by utilizing a nonreducing SDS-polyacrylamide gel electrophoresis/Western blot analysis. As shown in Figure 7A, the ebselen treatment resulted in the formation of a higher molecular weight Keap1-containing complex with an apparent molecular mass of greater than 150 kDa. This complex was not detected in the reducing SDS-polyacrylamide gel electrophoresis, suggesting that this form might correspond to an intermolecular disulfide-linked Keap1-containing complex. The ebselen-induced formation of the Keap1 complex was also confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis under nonreducing conditions in the first dimension and under reducing conditions (DTT) in the second dimension (Figure 7B). Among the phase 2-inducing electrophiles tested, the formation of this Keap1-containing complex was most prominently induced by ebselen in RL34 cells (Figure 7C) and in wild-type Keap1-overexpressed RL34 cells (Supporting Information, Figure S5). We also analyzed the in vitro modified recombinant Keap1 with ebselen (Supporting Information, Figure S6) and realized that, in comparison to the formation of the single peak of a higher molecular weight Keap1 complex generated in the ebselen-treated RL34 cells, ebselen modification


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Figure 5. Detection of biotin-cysteine-labeled (S-oxidized) proteins in RL34 cells treated with ebselen. (A) Chemical structure of biotinylated cysteine (biotin-cysteine). (B) Mechanisms for the formation of a biotin-cysteine-protein conjugate upon reaction of ebselen with the intra- and intermolecular disulfide linkages and selenenylsulfide linkage. (C) Two-dimensional gel electrophoresis of biotin-cysteine-labeled proteins. RL34 cells were treated with 100 µM biotin-cysteine for 30 min and then incubated with (panels a and b) or without (panels c and d) 20 µM ebselen for 1 h. The proteins were separated by isoelectrofocusing (pH range pH 3-10) and then by SDS-PAGE. All runs were carried out under nonreducing conditions. Panels a and b, SYPRO Ruby fluorescence staining. Panels c and d, Western blot. The red arrowheads denote spots excised for subsequent identification by MALDI-TOF analysis.

of the protein in vitro is rather random. Thus, it is likely thatthe system is much more finely tuned in vivo than those used for the in vitro studies.

C151 Is Required for the Formation of Keap1 Complex and Induction of Phase 2 Response by Ebselen. It has been proposed that phase 2 inducers disrupt the Keap1-Nrf2 complex



Table 1. Summary of Ebselen-Sensitive Proteins Identified spot no.

protein name

MW

1 2 3 4 5 6 7 8 9 10,11 12 13 14 15 16 17

heat shock protein 70 chaperonin (HSP60) vimentin Tuv-like 2 β-tublin enolase I unidentified proteasome 26S subunit ATPase 6 serine proteinase inhibitor, claide H glyseraldehyde 3-phosphate dehydrogenase laminin receptor 1 β-actin malate dehydrogenase prohibitin unidentified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein unidentified unidentified HSP27 peptidylprolyl isomerase

70827 60927 53700 51115 49963 47098

18, 19 20 21

45768 46488 36054 32803 41321 36461 29802 29103

22893 20295

by modifying the cysteine residues of Keap1 (27), allowing Nrf2 to translocate to the nucleus where, in heterodimeric combinations with other basic leucine zipper proteins, it binds to the ARE of the phase 2 genes and accelerates their transcription. To test the involvement of the Keap1 cysteine residues in the formation of the complex following exposure to ebselen, a number of mutant Keap1 genes containing alanine and serine codons substituted for specific cysteine codons were utilized. In the experiments reported in this study, we focused our attention on three cysteine residues (C151, C273, and C288) of Keap1. These three cysteine residues were selected on the basis of the recent reports that they preferentially react with thiolspecific chemicals in vitro (29, 36). As shown in Figure 8A, the ebselen-induced formation of high molecular weight species of Keap1 was rarely affected by transfection of cells with C273A and C288S Keap1, whereas overexpression of the C151S mutant protein almost completely suppressed the complex formation. Furthermore, substitution of C151 with a serine residue resulted in constitutive repression of the induction of class R GST A1 and class π GST P1 isozymes upon exposure to ebselen (Figure 8B). These data suggest that C151, and not C273 or C288, represents one of the target sites for modification by ebselen. This is consistent with the recent finding that C151 is one of the most readily modified cysteine residues by biotinylated iodoacetamide in Keap1 (46).

Discussion It has been well-documented that ebselen has a glutathione peroxidase (GPx)-like activity, which catalyzes the reduction of H2O2 and smaller organic hydroperoxides, including membrane-bound phospholipid and cholesterylester hydroperoxides. In addition, because ebselen is an effective antioxidant against membrane hydroperoxides, it inhibits both nonenzymatic and enzymatic lipid peroxidations in cells. In the meantime, ebselen has been shown to be a Trx peroxidase, which, in the presence of Trx, acts as a mimic of a peroxiredoxin (41). In the present study, we showed that ebselen dramatically down-regulated the spontaneous and HNE-stimulated productions of intracellular reactive oxygen species (ROS) in RL34 cells (Figure 2). On the basis of the previous kinetic study, this antioxidant function against intracellular oxidative stress may be ascribed to its peroxidase activity operating via the Trx system rather than GSH (38). It has been proposed that the ebselen mechanism of action as an H2O2 reductase mainly starts through the formation of

Figure 6. Effect of ebselen on the induction of phase 2 detoxification enzymes in RL34 cells. (A) Induction of phase 2 detoxification enzymes by ebselen. The cells were treated with ebselen (10 µM) for different time intervals as indicated in the figures. RT-PCR was performed using total RNA isolated from RL34 cells treated with ebselen. (B) Activation of ARE/EpRE reporter genes by ebselen. Either pGSTA1-Luc, pHO1-Luc, pQR-Luc, or pRBGP3 reporter plasmid was transfected into RL34 cells. After transfection, the medium was changed to fresh medium and the cells were treated with DMSO (open bar) or 10 µM ebselen (closed bar). The vertical bars represent SD values of triplicate determinations. (C) Nuclear accumulation of Nrf2 in the RL34 cells treated with ebselen (10 µM).

the ebselen selenol. This reduced form of ebselen quickly reacts with H2O2 to form H2O and the ebselen selenenic acid, which spontaneously releases another molecule of H2O and regenerates ebselen, thus finishing a catalytic cycle (Figure 3). In the presence of Trx, the formation of the ebselen selenol can occur both directly via TrxR and by means of the reduced Trx. Thus, the antioxidant action of ebselen against intracellular ROS production may be, to a large extent, due to reactions with the Trx system. Despite the potential antioxidant effect against intracellular ROS production, ebselen is known to be a highly efficient oxidant of Trx (41). To establish the mechanism for modification of Trx by ebselen, human recombinant Trx was incubated with ebselen and analyzed by SDS-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses. The nonreducing SDS-polyacrylamide gel electrophoresis indeed showed that ebselen is a chemically reactive species capable of converting Trx to the higher molecular weight protein species corresponding to the intermolecular cross-linking reactions (Figure 4). The MADLTOF MS analysis of the ebselen-treated Trx indeed detected the peak corresponding to the intermolecular disulfide linkage at C73 (Supporting Information, Figure S2) and the peaks


Sakurai et al.

Figure 8. Requirement of C151 for the formation of Keap1 complex and induction of phase 2 response by ebselen. (A) RL34 cells transfected with expression vectors for either wild-type (WT) or the indicated mutant Keap1 proteins were treated with or without 20 µM ebselen for 1 h. Cell lysates were collected, electrophoresed through an SDSpolyacrylamide gel, and subjected to immunoblot analysis with an antibody against Keap1. The arrows on the right side of the figure indicate the location of Keap1 and of an ebselen-induced form of Keap1 that migrates with retarded mobility through an SDS-polyacrylamide gel. (B) Effect of transient overexpressions of WT and mutant Keap1 upon induction of GST by ebselen. Transient transfections of RL34 cells were performed with either WT or mutant Keap1 constructs, as described under the Materials and Methods. Empty vectors were used as the control transfection experiment. Twenty-four hours after transfection, the RL34 cells were treated with or without 20 µM ebselen for 24 h, and then, the cell lysates were subjected to immunoblot analysis for GST A1 and GST P1. The results are representative of three separate experiments.

Figure 7. Ebselen-induced formation of a Keap1 complex in RL34 cells. (A) Detection of Keap1 in RL34 cells treated with ebselen. RL34 cells were incubated with 50 µM ebselen for 1 h. Cell lysates were collected, treated with or without 50 mM DTT, electrophoresed through an SDS-polyacrylamide gel, and subjected to immunoblot analysis with an antibody against Keap1. (B) Detection of modified Keap1 by 2D SDS-polyacrylamide gel electrophoresis. Panels a and b, SyproRuby staining. Panels c and d, immunoblot analysis using anti-Keap1 antibody. The Keap1 complex was analyzed by 2D SDS-polyacrylamide gel electrophoresis under nonreducing conditions in the first dimension and under reducing conditions (2-mercaptoethanol) in the second dimension. (C) Detection of Keap1 in RL34 cells treated with electrophiles. RL34 cells were incubated with an electrophile (10 or 50 µM) for 1 h.

corresponding to the intramolecular disulfide linkages between C32 and C35 (Supporting Information, Figure S3) and to the intramolecular disulfide linkages between C62 and C69 (Supporting Information, Figure S4). Detection of a mass shift indicative of the addition of one molecule of ebselen per protein at C73 (Supporting Information, Figure S1) suggest the proposed mechanism, in which the initially formed selenenylsulfide between ebselen and Trx can be rapidly reduced by another highly reactive cysteine thiol of the protein to form a disulfide

linkage (Figure 4C). Thus, Trx may be randomly oxidized at five of the cysteine residues in Trx and the preference for the oxidative modification of the catalytic cysteines (C32 and C35) to noncatalytic cysteines (C62, C69, and C73) was not observed. The cysteine-targeted oxidation followed by S-thiolation is an oxidative, reversible post-translational modification of the 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 the cellular redox status and has no absolute requirement for specialized regulatory enzymes, although thiol transferase enzymes can catalyze these reactions (47). 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 (48, 49). In particular, the protein S-thiolation/dethiolation is a dynamic process that occurs in cells under physiological conditions as well as following exposure to an oxidative stress (50-52). 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) (51). To investigate the electrophilic potential of ebselen, we analyzed the S-oxidized proteins in the cells after exposure to ebselen (Figure 5). To this end, we utilized biotin-cysteine as the probe in the investigation of the protein S-oxidation. 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-tiolated proteins could be detected on nonreducing Western blots using streptavidin-HRP, and these were quantified via digitization. They could also be purified using a streptavidin


affinity matrix and then identified using Western immunoblotting. The proteomics identification of the S-thiolated proteins revealed that ebselen induced S-thiolation of proteins, including cytoskeletal proteins, glycolytic enzymes, and chaperone protein (Figure 5C 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. The majority of the phase 2 inducers appear to have a common chemical featuresthe ability to react with sulfhydryl groups. Because of its high cysteine content, Keap1 has been suggested to be an excellent candidate as the sensor for phase 2 inducers (45). Taking advantage of the unique electrophilic property of ebselen giving rise to the formation of selenenylsulfide and disulfide linkages within proteins without generating ROS, we characterized the oxidative modification of Keap1 in ebselen-treated cells and found that ebselen induced modification of Keap1 by generating a higher molecular weight Keap1containing complex (Figure 7). Disappearance of the modified form of Keap1 under reducing conditions during electrophoresis through SDS-polyacrylamide gels indicates that the modified Keap1 protein is a disulfide-linked complex. A similar modified form of Keap1 was also detected in the cells treated with other known inducers of the ARE-dependent gene expression (Supporting Information, Figure S5). Our observations that (i) formation of the modified form of Keap1 is abolished by the C151S substitution (Figure 8A) and (ii) the C151 mutation also resulted in constitutive repression of the induction of class R GST A1 and class π GST P1 isozymes upon exposure to ebselen (Figure 8B) suggest a link between the posttranslational modification to C151 of Keap1 and the induction of the phase 2 response. This proposition may be supported by the previous findings that C151 is uniquely required for inhibition of the Keap1-dependent degradation of Nrf2 by phase 2 inducers (25) and that C151 is one of the most reactive cysteine residues in Keap1 to thiol-reactive chemical inducers (29, 46). It has been proposed that this C151 modification by inducers may alter the conformation of the Keap1 BTB domain by altering the homodimerization interface, and this structural alteration of BTB domain may simultaneously result in the complex formation and ubiquitination of Keap1 (29). Thus, C151 may be critical not only for the ebselen sensing function of Keap1 but also for its repressor activity. On the other hand, other cysteine residues, such as C273 and C288, have also been shown to be critical for exposure to inducers leading to the formation of disulfidelinked dimers of Keap1 (27). A model has been proposed, in which modification of C273 and C288 in Keap1 by inducers directly causes dissociation of the Keap1-Nrf2 interaction, thereby promoting Nrf2 nuclear accumulation and hence ARE activation (27). However, the present study showed that both the complex formation and the phase 2 induction by ebselen were rarely affected by the individual mutation of C273 and C288 (Figure 8). Thus, sites of sulfydryl oxidation may vary among the different classes of inducers. It might be possible that different reagents and reaction conditions could be sensed by different cysteines. Further studies focusing on the cysteinetargeted oxidation of Keap1 by ebselen would extend our understanding of the regulation of the phase 2 detoxification cascades stimulated by other inducers. Acknowledgment. We are grateful to Dr. Junji Yodoi of Kyoto University for his kind gift of recombinant Trx. This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology and by the Center of Excellence (COE) Program in the 21st Century


in Japan (K.U.), by research fellowships of the Japan Society for the Promotion of Science (T.S.), by a research grant from the Institute for Advance Research, Nagoya University (K.U.), and by the COE Program in the 21st Century in Japan (K.U.). Supporting Information Available: MALDI-TOF MS analysis of the trytptic fragments from native ebselen-treated Trx, electrophile-induced formation of a Keap1 complex in RL34 cells, and oxidative modification of Keap1 by ebselen. This material is available free of charge via the Internet at http://pubs.acs.org.

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Ebselen, a Seleno-organic Antioxidant, as an Electrophile - American

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