Catechol Type Polyphenol Is a Potential Modifier of Protein Sulfhydryls

Oct 9, 2009 - of Shizuoka, Shizuoka 422-8526, Japan, Department of Biological ... Sciences, Osaka Prefecture UniVersity, Sakai 599-8531, Japan, and...
1 downloads 0 Views 2MB Size
Chem. Res. Toxicol. 2009, 22, 1689–1698

1689

Catechol Type Polyphenol Is a Potential Modifier of Protein Sulfhydryls: Development and Application of a New Probe for Understanding the Dietary Polyphenol Actions Takeshi Ishii,†,‡ Miki Ishikawa,† Noriyuki Miyoshi,†,‡ Mayuko Yasunaga,† Mitsugu Akagawa,§ Koji Uchida,† and Yoshimasa Nakamura*,†,| Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan, Department of Food and Nutritional Sciences, and Global COE Program, UniVersity of Shizuoka, Shizuoka 422-8526, Japan, Department of Biological Chemistry, DiVision of Applied Life Science, Graduate School of Life and EnVironmental Sciences, Osaka Prefecture UniVersity, Sakai 599-8531, Japan, and Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama UniVersity, Okayama 700-8530, Japan ReceiVed April 22, 2009

The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, β-actin. This identification was confirmed by the pull-down assay with anti-β-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous β-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified β-actin. These results indicate that β-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification. Introduction Dietary polyphenols, such as flavonoids, are the most common and widely distributed phytochemicals in fruits and vegetables, including green tea, wine, cocoa, and berries (1). The antioxidant properties of these compounds are often claimed to be responsible for the protective effects of these components against cardiovascular disease, cancer, and/or many other diseases (2). In addition, it has been proposed that the accumulation of oxidative damage is an important contributor to not only pathological conditions but also the aging process (3). Therefore, the antioxidant action is believed to have beneficial health effects on diseases and aging. Indeed, some of these beneficial effects have been demonstrated in animal models and in some epidemiological studies (2, 4). Nevertheless, only some of these activities have been demonstrated to be associated with the antioxidative activities of polyphenolic flavonoids (5). Alter* To whom correspondence should be addressed. Tel: +81-86-251-8300. Fax: +81-86-251-8388. E-mail: [email protected]. † Nagoya University. ‡ University of Shizuoka. § Osaka Prefecture University. | Okayama University.

natively, a wealth of data exists that suggests that most of the relevant mechanisms of disease prevention by polyphenolic flavonoids, such as in cancer, are not related to their antioxidant properties but rather are due to the prooxidant action and the direct interaction of flavonoids and target molecules (6, 7). Searching for the dietary polyphenol “sensor” or high affinity proteins that bind to polyphenolic flavonoids is the first step in understanding the molecular and biochemical mechanisms of the functional effects of dietary polyphenols. A few proteins that can directly bind with flavonoids have been identified by predicting the target molecule, including fibronectin, fibrinogen, histidine-rich glycoprotein, fatty acid synthase, serum albumin, and laminin receptor (8-11). Recently, laminin receptor was reported as a catechin receptor that mediates the anticancer activity of (-)-epigallocatechin gallate (EGCg)1 (12). In addition, vimentin was identified as a novel molecular target of the EGCg-protein interaction by affinity chromatography using an EGCg column (13). However, the biological and physiological significance of the functional effects of polyphenolic flavonoids is not clear due to the existence of other targets, and the binding structure remains unidentified.

10.1021/tx900148k CCC: $40.75  2009 American Chemical Society Published on Web 09/10/2009

1690

Chem. Res. Toxicol., Vol. 22, No. 10, 2009

Several lines of evidence indicate that the catechol type polyphenols have poor stability in neutral or alkaline solution (14). Moreover, previous reports showed that the catechol type polyphenols were easily oxidized in a cell culture medium with slightly alkaline pH (15). Oxidation of flavonoids with a catechol structural motif in their B ring leads to the formation of a flavonoid quinone, which rapidly reacts with sulfhydryls in GSH or protein cysteine residues to form cysteinyl flavonoid adducts (16, 17). More recently, we found that EGCg forms covalent adducts with protein sulfhydryls through autoxidation (18). Many studies have implicated cysteine sulfhydryls present in various transcription factors, such as kelch-like ECH associated protein 1/nuclear respiratory factors 1 (Keap1/Nrf2), nuclear factor-κB (NF-κB), and p53 as redox sensors for the transcriptional regulation of many genes essential for maintaining cellular homeostasis. Some chemopreventive and cytoprotective agents have been found to target cysteine sulfhydryls present in key transcription factors or their regulators, thereby suppressing the aberrant overactivation of carcinogenic signal transduction or restoring/normalizing or even potentiating cellular defense signaling (19-21). In the present study, to investigate the protein sulfhydryl modification by the catechol type polyphenols, we used 3,4dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect the protein modification by polyphenol compounds using a biotintagged DPA (Bio-DPA). Thus, a model protein, glyceraldehyde3-phosphate dehydrogenase (GAPDH), was exposed to DPA or Bio-DPA, and the modification behavior was characterized using Western blotting and MS. In addition, RL34 cells were treated with Bio-DPA, and the target proteins were identified using a proteomic approach.

Materials and Methods Materials. DPA, laccase, RNase from bovine, angiotensin Ι, EC, ECg, and quercetin were obtained from Sigma-Aldrich (St. Louis, MO). GAPDH was purchased from Roche Diagnostics (Basel, Switzerland). Horseradish peroxidase (HRP)-linked antirabbit IgG, HRP-conjugated NeutrAvidin (HRP-avidin), Cy5-labeled avidin, Protein G-Sepharose, normal goat serum, and enhanced chemiluminescence (ECL) Western blotting detection reagents were purchased from GE Healthcare UK Ltd. (Buckinghamshire, United Kingdom). EZ-link 5-(biotinamido) pentylamine, sulfo-N-hydroxysuccinimide, and 1-ethyl-3-(dimethylaminopropyl) carbodiimide were obtained from Thermo Fisher Scientific, Inc. (San Jose, CA). The anti-β-actin polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sequence grade modified trypsin was obtained from Promega Co. (Madison, WI). Calmodulin-dependent protein kinase II 281-289 (CaMKII, Seq.: MHRQETVDC), β-lactoglobulin (β-LG) from bovine milk, EDTA, dithiothreitol (DTT), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), GSH, iodoacetoamide, trifluoroacetic acid (TFA), (-)-epicatechin (EC), and (-)-epicat1 Abbreviations: ARE, antioxidant response element; β-LG, β-lactoglobulin; BSA, bovine serum albumin; CaMKII, calmodulin-dependent protein kinase II; DPA, 3,4-dihydroxyphenyl acetic acid; Bio-DPA, biotin-tagged DPA; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid); DTT, dithiothreitol; ECL, enhanced chemiluminescence; EC, (-)-epicatechin; ECg, (-)-epicatechin gallate; EGCg, (-)-epigallocatechin gallate; ESI, electrospray ionization; FBS, fetal bovine serum; F-actin, filamentous actin; G-actin, globular actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSTP1, glutathioneS-transferase subunit-P1; HRP, horseradish peroxidase; HRP-avidin, HRPconjugated NeutrAvidin; Keap1, kelch-like ECH associated protein 1; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NF-κB, nuclear factor-κB; NMR, nuclear magnetic resonance; NQO1, quinone oxidoreductase 1; Nrf2, nuclear respiratory factors 1; PMF, peptide mass fingerprints; ROS, reactive oxygen species; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TOF, timeof-flight.

Ishii et al. echin gallate (ECg) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GSH Assay. Measurement of the GSH level was performed spectrophotometrically using commercial kit GSH-400 (Oxis International Inc., Foster City, CA) (22). GSH (100 µM) was incubated with 0-100 µM DPA in 100 mM sodium phosphate buffer (pH 7.4) at 25 °C for 1 h with or without laccase (30 units), with gentle agitation in the dark. Fifty microliters of 12 mM chromogenic reagent in 0.2 M HCl was added to the reaction mixture (300 µL) at the end of incubation period and mixed thoroughly. After 50 µL of 7.5 M NaOH was added and mixed, the mixture was incubated at 25 °C for 10 min, and then, absorbance was determined spectrophotometrically at 400 nm. Detection and characterization of GSH-DPA adducts were performed by both electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC MS; see below) and nuclear magnetic resonance (NMR) spectrometry using a Bruker AMX400 (400 MHz; Bruker Daltonics, Ltd., Karlsruhe, Germany). Matrix-Assisted Laser Desorption Ionization-Time-ofFlight Mass Spectrometry (MALDI-TOF MS). The sulfhydrylcontaining peptide (MHRQETVDC, 0.1 mg/mL) was incubated with 0 or 100 µM DPA for 1 h at 37 °C in a buffer containing 50 mM phosphate buffer (pH 7.5) with or without laccase (30 units) and then mixed with TFA. To improve the ionization efficiency of MS, samples were purified with Zip Tip µ-C18 (Millipore, Bedford, MA) before MALDI-TOF MS analysis. Peptides were mixed with 2.5 mg/mL R-cyano-4-hydroxycinnamic acid (R-CHCA) containing 50% acetonitrile and 0.1% TFA and dried on stainless steel targets at room temperature and pressure. The analyses were performed using an UltraFLEX MALDI-TOF MS (Bruker Daltonics, Ltd., Bremen, Germany). All analyses were carried out in the positive ion mode, and the instrument was calibrated immediately prior to each series of studies. ESI-Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The peptide samples were analyzed by reversedphase HPLC, a system that consisted of a nanospace SI-1 HPLC system (Shiseido Co., Ltd., Tokyo, Japan), using a Capcell Pak C18 UG120 column (2.0 mm × 250 mm i.d.; Shiseido). These samples were eluted with a linear gradient of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) (time ) 0-3 min, 10% B; 3-45 min, 10-40% B; 45-50 min, 40-50% B; 50-52 min, 50-80% B). The flow rate was 0.2 mL/min, and the column temperature was controlled at 40 °C. The MS (MS/MS) analyses were performed on an LCQ ion trap mass system (Thermo Fisher Scientific, Inc.) equipped with an electrospray ion source. The electrospray system employed a 5 kV spray voltage and a capillary temperature of 260 °C. Collisioninduced dissociation experiments in the positive ion mode were performed by setting the relative collision energy at 30% using helium as the collision gas. Preparation of Bio-DPA. The carboxyl group of DPA was modified by amidation with EZ-link 5-(biotinamido) pentylamine by a modification of a previously described procedure (23). The Bio-DPA was purified through a reverse-phase HPLC eluted with a linear gradient of acetonitrile/water/TFA. The bio-DPA was then dried under argon and dissolved in Me2SO for further use. Reaction of GAPDH with Bio-DPA. The GAPDH solution (1 mg/mL) was incubated with 0-100 µM Bio-DPA in 50 mM sodium phosphate buffer (pH 7.2) with or without laccase (30 units) at 37 °C for 1 h. The reaction was terminated by centrifugal filtration (Microcon 30, molecular weight cutoff of 30000; Millipore Co., Billerica, MA) to remove the low molecular weight reactants. Control experiments were performed under the same conditions without reaction of laccase. DTNB Assay. Loss of sulfhydryl groups in GAPDH was assayed as previously reported (24). An aliquot (0.1 mL) of the protein samples (1 mg/mL) was mixed with an equal volume of 20% trichloroacetic acid (TCA, w/v) and centrifuged at 12000g for 10 min at 4 °C. The pellet was washed twice with ethanol-ethyl acetate (1:1 v/v), and the pellet was then dissolved with 0.4 mL of 8 M guanidine hydrochloride, 13 mM EDTA, and 133 mM Tris

Catechol Type Polyphenol May Modify Protein Sulfhydryls solution (pH 7.6). DTNB (45 µL of 10 mM solution) was then added, and the color was allowed to develop for 5 min. The absorbance was spectrophotometrically measured at 412 nm. The values were calculated by comparison with the identically treated N-acetylcysteine standards (10-300 µM). ELISA. A 100 µL aliquot of the sample solution (1 mg/mL) was added to each well of a 96 well microtiter plate and incubated for 1 h at 37 °C. The sample was then removed, and the plate was washed with PBS containing 0.05% Tween 20 (PBS-T) and water. Each well was filled with Block Ace solution (40 mg/mL) for 1 h at 37 °C. HRP-avidin 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 the wells were washed three times with PBS-T, HRPavidin 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 0.03% 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 Laboratories, Inc., Hercules, CA). Dot Blot and Western Blot Analyses. For the detection of DPAmodified proteins, GAPDH and whole cell lysates from RL34 cells were treated with sodium dodecyl sulfate (SDS) sample buffer with reducing agent for 5 min at 100 °C. The samples were then transblotted or separated by 10 or 12.5% SDS-polyacrylamide gel electrophoresis (PAGE). The gel was transblotted onto a nitrocellulose or PVDF membrane. The membranes were incubated with 5% skim milk for blocking, washed, and incubated with the HRPavidin. This procedure was followed by the addition of ECL reagents. The bands and spots were visualized by Cool Saver AE6955 (ATTO, Tokyo, Japan). Comparison of Modification Behavior of Bio-DPA to Proteins. Thirty micromolar protein samples (GAPDH, β-LG, and RNase) were incubated with 0-100 µM Bio-DPA in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with laccase. The reaction was terminated by centrifugal filtration (Microcon 10, molecular weight cutoff of 10000) to remove the low molecular weight reactants. Bio-DPA-modified proteins were detected by Western blot analysis with HRP-avidin, followed by the addition of ECL reagents. The bands were visualized by Cool Saver AE6955 in same exposure condition. Cell Culture. RL34 cells were obtained from the Japanese Cancer Research Resources Bank. The cells were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, 588 µg/mL L-glutamine/L-Gln, and 0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2. 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 Cy5-labeled avidin, rinsed with PBS containing 0.3% Triton X-100, and covered with antifade 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). Peptide Mass Fingerprints (PMF). PMF was performed as previously reported (11). Briefly, peptide extracts from gel pieces were analyzed with an UltraFLEX MALDI-TOF MS (Bruker Daltonics, Ltd.). A few microliters of the sample was mixed with equal volumes of a saturated solution of sinapinic acid or R-cyano4-hydroxycinnamic acid (Bruker Daltonics) in acetonitrile/0.1% TFA, and the mixture (1 µL) was deposited on the MALDI-TOF MS target. Proteins were identified with MASCOT (Matrix Science, London, United Kingdom) searching algorithms using the nonredundant database. Pull-Down and Immunoprecipitation Assays. RL34 cells incubated with and without 50 µM Bio-DPA in a control medium for 1 h were washed with PBS, harvested, and lysed in 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% NP-40, and

Chem. Res. Toxicol., Vol. 22, No. 10, 2009 1691 protease inhibitors. Cell lysates containing 0.5 mg of protein were incubated batchwise with 25 µL of StreptAvidin-Plus beads overnight at 4 °C with constant shaking. The beads were rinsed five times with lysis buffer by centrifugation at 3000 rpm for 5 min. The proteins were eluted by boiling the beads in SDS-sample buffer for 5 min and analyzed by SDS-PAGE followed by immunodetection with anti-β-actin polyclonal antibody. In addition, the cell lysates were incubated with 2 µg of anti-β-actin polyclonal antibody overnight at 4 °C. The mixture was then treated with 20 µL of Protein G-Sepharose and incubated for 1 h at 4 °C. The mixture was then centrifuged (3000 rpm, 5 min), rinsed five times with lysis buffer, and subsequently boiled with SDS-sample buffer. The biotinylated proteins were then subjected to immunoblot and detection with HRP-avidin and ECL.

Results Modification Behavior of DPA to Sulfhydryls. To investigate the modification behavior of catechol type polyphenols on intracellular sulfhydryls such as GSH and protein, we used DPA as a model catechol compound (Figure 1A). Previous reports indicate that the incubation of the catechol type polyphenol with polyphenoloxidase, an enzyme capable of catalyzing the two-electron oxidation of catechol moieties (25), in the presence of the sulfhydryls, results in the formation of the polyphenol adducts (16). We first examined the modification behavior of DPA with GSH, which is the most abundant intracellular sulfhydryl, in the presence of laccase. As shown in Figure 1B, incubation of 0.1 mM GSH with DPA (100 µM) or laccase (30 units) alone for 1 h resulted in no significant GSH loss. On the other hand, the coincubation of both DPA and laccase with GSH resulted in the complete loss of GSH (Figure 1 B). Approximately 60% of GSH was lost after a 1 h incubation with 20 µM DPA (Figure 1C), suggesting that DPA not only reacted with GSH but also oxidized GSH into GSSG possibly due to the generation of reactive oxygen species (ROS) accompanied by the formation of DPA-quinone. To confirm the formation of the GSH-DPA adduct, the reaction mixture with laccase was analyzed by ESI-LC-MS. As shown in Figure 1D, a new peak was detected on the HPLC chromatogram by UV (215 nm) detection. As expected, the molecular ion peak at m/z 474.0, which corresponded to the GSH-DPA adduct, was detected at the same UV peak retention time (Figure 1D). In addition, this major adduct was identified as 6-glutathionyl-DPA by its NMR spectral data (Figure 1E). These results indicate that DPA reacts with GSH through the formation of an o-quinone structure accompanied by the oxidation of the catechol moiety. Subsequently, to gain further details about the sulfhydryl modification by DPA, we used both the cysteine peptide CaMKII (MHRQETVDC) and its S-carbamidomethyl derivative as the model peptides (Figure 2A). MALDI-TOF MS analysis of the native peptide gave a molecular mass of m/z ) 1118.4, which was in agreement with the theoretical molecular mass (Figure 2Ba, lower). When the peptide was incubated with 100 µM DPA for 1 h at 37 °C in the presence of laccase, the DPAmodified peptide (m/z ) 1270.8) was significantly detected by MALDI-TOF MS analysis (Figure 2Ba, upper). However, the reactivity remarkably decreased by the S-carbamidomethylation of the cysteine residue (Figure 2Bb, upper). This suggests that the modification site of DPA was a cysteine residue in the peptide. To clarify the DPA modification sites in this peptide, the native and DPA-treated peptides were further analyzed by ESI-LC-MS/MS. The MS/MS spectrum of the [M + H]+ at m/z ) 1270.5 from the DPA-modified peptide is shown in Figure 2C. In the MS/MS analysis, the singly charged N-terminal

1692

Chem. Res. Toxicol., Vol. 22, No. 10, 2009

Figure 1. Modification behavior of DPA to GSH. (A) Chemical structure of DPA. (B) GSH consumption by DPA. GSH (100 µM) was incubated with or without DPA (100 µM) in 100 mM sodium phosphate buffer (pH 7.4) at 25 °C for 1 h in the presence or absence of laccase (30 units). The amount of residual GSH was spectrophotometrically estimated using the commercial kit GSH-400. (C) Dose-dependent GSH consumption by DPA. GSH (100 µM) was incubated with 0-100 µM DPA in 100 mM sodium phosphate buffer (pH 7.4) at 25 °C for 1 h with or without laccase. The amount of residual GSH was spectrophotometrically estimated using the commercial kit GSH-400. (D) Detection of GSH-DPA adducts by LC-MS analysis. GSH (100 µM) was incubated with DPA in 100 mM sodium phosphate buffer (pH 7.4) at 25 °C for 1 h in the presence of laccase. The LC-MS measurements were performed by monitoring ions at abs. 215 nm (lower) and m/z 478 (upper). (E) 1H NMR analysis of GSH-DPA adduct. A mixture of DPA and DPA-GSH was dissolved in D2O and then analyzed by NMR spectrometry.

product ions (b3-b8) and H2O loss fragment ions (b4-18, b7-18, and b8-18) were observed. Fragment ions (y2, y3, y6, y7, and y8) and the H2O loss fragment ion (y4-18) were observed to increase by 152 Da. In addition, neutral loss ions ([M + H - 152]+) were also observed. These results indicate that the DPA modification site in the peptide is a cysteine residue. Thus, using DPA and the peptides containing a sulfhydryl, we showed the high reactivity and selectivity for the cysteine residues of the catechol compound. Detection of the Protein Modification by Bio-DPA. Several analytical approaches involving affinity purification of polyphenol binding proteins have been reported (8-13, 18). However, because of the use of cell lysates, these approaches do not provide direct evidence for the binding with intracellular proteins. Biotinylation reagents are available for targeting a variety of specific functional groups, including primary amines, carboxylates, and sulfhydryls. Typical flavonoids such as

Ishii et al.

Figure 2. Modification behavior of DPA to CaMKII. (A) Sequences of CaMKII (a) and its S-carbamidomethyl derivative (b). (B) MALDITOF MS analysis of DPA-treated peptides. CaMKII (a, 0.1 mg/mL) or S-carbamidomethylated CaMKII (b, S-blocked CaMKII, 0.1 mg/ mL) were incubated with DPA (100 µM) in the presence (upper) or absence (lower) of laccase at 37 °C for 1 h. (C) MS/MS analysis of a CAMKII-DPA adduct.

quercetin and catechins, lacking of these functional groups besides hydroxyl groups, are insufficient for biotinylation. In addition, we previously demonstrated that protocatuchuic acid, a simple catechol compound, is converted into the reactive quinone intermediate(s) by phenol oxidase, which can bind nucleophilic residues of proteins and alter the cellular immune functions (26). This study supported the idea that a simple catechol structure is the minimum determinant for protein modification by polyphenol compounds. Therefore, the preliminary study was undertaken to make a biotin probe using protocatechuic acid, which has a carboxyl group bonded directly to benzene ring. However, the reactivity of protocatechuic with the biotinylation reagent is too low to provide the desired compounds efficiently. Therefore, in this study, we used DPA as a model catechol compound containing a carboxyl group, which has a good reactivity with the biotinylation reagent. To directly detect the protein modification by catechol type polyphenols, Bio-DPA was synthesized (Figure 3A), and the modification behavior was characterized by applications that exploit the specificity of the avidin-biotin complex. Hence, we examined the potential reactivity of Bio-DPA toward sulfhydryl enzymes, using GAPDH as a convenient model protein that contains four sulfhydryl groups per subunit and is known to be

Catechol Type Polyphenol May Modify Protein Sulfhydryls

Chem. Res. Toxicol., Vol. 22, No. 10, 2009 1693

Figure 3. Covalent binding of Bio-DPA to protein sulfhydryls. (A) Chemical structure of Bio-DPA. (B) Loss of sulfhydryl groups in GAPDH. GAPDH (1 mg/mL) was incubated with 50 µM Bio-DPA in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with or without laccase. After the incubation, an aliquot (0.1 mL) was taken from the reaction mixture, and the amount of sulfhydryl groups was determined by DTNB assay as described in the Materials and Methods. (C) ELISA of Bio-DPA-modified GAPDH probed with HRP-avidin. GAPDH (1 mg/mL) was incubated with 0-100 µM Bio-DPA in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with (b) or without (2) laccase. (D) Western blot analysis of Bio-DPA-modified GAPDH probed with HRP-avidin. GAPDH (1 mg/mL) was incubated with 0-50 µM Bio-DPA in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with (left panel) or without (right panel) laccase. (E) Modification behavior of Bio-DPA to GAPDH in the presence of a protein sulfhydryl alkylating agent. GAPDH (1 mg/mL) was incubated with 50 µM Bio-DPA and laccase in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with (upper panel) or without (lower panel) iodoacetoamide. (F) Modification behavior of Bio-DPA to proteins. The protein samples (GAPDH, β-LG, and RNase) were incubated with 0-100 µM Bio-DPA in 100 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min with laccase. Bio-DPA-modified proteins were detected by Western blot analysis with HRP-avidin.

highly sensitive to modification by electrophiles in vitro (24, 25, 27). As shown in Figure 3B, upon incubation of GAPDH (1 mg/mL) with 50 µM Bio-DPA in the presence of laccase, the free sulfhydryl groups of GAPDH were remarkably decreased. In addition, the Bio-DPA-induced loss of the free sulfhydryl groups was accompanied by an increase in ELISApositive rates probed with an HRP-avidin, due to the binding of Bio-DPA (Figure 3C). To evaluate the binding of Bio-DPA to the sulfhydryl groups of the enzyme, we examined the incorporation of Bio-DPA to the enzyme with or without laccase by SDS-PAGE. In the presence of laccase, sulfhydryl modification by Bio-DPA was confirmed by HRP-avidin blot analysis (Figure 3D, left panel). However, the modification can be barely observed without laccase (Figure 3D, right panel), suggesting that the oxidation of the catechol moiety on Bio-DPA is important for protein modification. Moreover, the incorporation of Bio-DPA was significantly inhibited by coincubation with iodoacetoamide, which is an alkylating agent of protein sulf-

hydryls (Figure 3E). This result strongly suggests that Bio-DPA exclusively binds to the cysteine residues of GAPDH to form the covalent binding. In an attempt to gain support for our hypothesis that the protein modification by Bio-DPA might be achieved through the direct electrophile scavenger function of the sulfhydryl groups, we compared the Bio-DPA incorporating rate of GAPDH (contains four sulfhydryl groups per subunit) with other proteins, such as β-LG (contains a sulfhydryl group per subunit) and bovine RNase (contains no sulfhydryl group per subunit). The Bio-DPA incorporating rate was assessed by an HRP-avidin blot analysis at various concentrations during the incubation. As shown in Figure 3F, among the proteins tested, GAPDH showed the highest incorporating rate of BioDPA. In contrast to the remarkably fast rate of scavenging by GAPDH, Bio-DPA had still not been incorporated into other proteins at 10 µM. On the other hand, Bio-DPA was incorporated into β-LG and bovine RNase at 100 µM. Oxidation of polyphenols with a catechol structural motif leads to the

1694

Chem. Res. Toxicol., Vol. 22, No. 10, 2009

Ishii et al.

Figure 5. Modification of β-actin by Bio-DPA in RL34 cells. RL34 cells were treated with 50 µM Bio-DPA for 1 h. Cell lysates were incubated with Immobilized NeutrAvidin or with anti-β-actin, as indicated. The presence of the Bio-DPA-modified β-actin was detected by immunoblot analysis (upper panel), and the incorporation of BioDPA into the β-actin immunoprecipitates was detected with HRP-avidin and ECL (lower panel).

Figure 4. Detection of Bio-DPA-modified proteins in RL34 cells. (A) Immunocytochemical detection of Bio-DPA incorporation into the cells. RL34 cells were treated with 100 µM Bio-DPA for 1 h. (B) Dot blot analysis of Bio-DPA-protein adducts probed with HRP-avidin. (C) Western blot analysis of Bio-DPA-protein adducts probed with HRPavidin. In panels B and C, cells were treated with 50 µM Bio-DPA for 1 h. The protein samples were direct-transblotted or separated by SDSPAGE followed by Western blotting. (D) The cells were lysed with a sonicater in PBS with protease inhibitors. After separation by centrifuge, the supernatant was removed and reserved as the soluble cytoplasmic fraction. The precipitated pellet was then dissolved in SDS-sample buffer and kept as the insoluble fraction containing membranes, organelles, and cytoskeleton. Each protein sample was separated by SDS-PAGE followed by Western blotting.

formation of a polyphenol quinone, which rapidly reacts with sulfhydryls in GSH or protein cysteine residues to form cysteinyl polyphenol adducts. Although the reactivity is lower than that of sulfhydryl group, amino and imidazol moieties are targets of polyphenol quinone to form stable adducts (28). Bio-DPA may react with lysine and histidine residues in the proteins. These data suggest that the sulfhydryl groups may represent the direct sensors of the catechol type polyphenols. Identification of Target Protein of Bio-DPA in RL34 Cells. To detect cellular proteins that undergo cysteine-targeted modification through the oxidation of the catechol moiety, BioDPA, having the similar redox potential to DPA (data not shown), was utilized as a molecular probe. As shown in Figure 4A, when RL34 cells were treated with 100 µM Bio-DPA for 1 h, a significant incorporation of Bio-DPA into the cells was

observed. After exposure to Bio-DPA, the whole cell lysates were blotted onto a PVDF membrane, and the biotin-labeled proteins were analyzed by dot blot and Western blot analyses probed with HRP-avidin. When RL34 cells were treated with 0-100 µM Bio-DPA for 1 h, the biotin-labeled proteins were detected in a dose-dependent manner (Figure 4B). As shown in Figure 4C, the Bio-DPA-modified proteins were detected around 40 (p-1) and 70 kDa (p-2) when assayed by Western blotting. In addition, this 40 kDa band was prominently detected in the insoluble fraction containing membranes, organelles, and cytoskeleton (Figure 4D). For proteomic identification, these bands were excised from the SDS-PAGE gels, subjected to trypsin digestion, and analyzed by MALDI-TOF MS (data not shown). The p-1 was identified to be β-actin by PMF analysis. Using MASCOT, the probability-based MOWSE score was 126 for β-actin (p < 0.05). In addition, p-2 was identified as bovine serum albumin (BSA) by the analysis (data not shown), which is a contaminant from a cell culture medium with FBS. BioDPA can bind to BSA, since serum albumin is one of the major targets of interaction by polyphenols (11). On the other hand, GAPDH, reactive with DPA in vitro, was not detected as a BioDPA target, assuming that the biotin tag could sterically hinder the ability of DPA to access protein cysteine residues. We then attempted to detect the modification of β-actin in cells exposed to Bio-DPA. To this end, RL-34 cells were treated with 50 µM Bio-DPA for 1 h, and the cell lysates were incubated with NeutrAvidin beads. After washing with lysis buffer, proteins bound to the resin through Bio-DPA were eluted with SDS-PAGE sample buffer, and β-actin was detected by Western blot analysis using the anti-β-actin polyclonal antibody (Figure 5, upper panel). Alternatively, the cell lysates were subjected to immunoprecipitation with the antibody, and the presence of Bio-DPA-modified proteins was detected by Western blotting probed with HRP-avidin (Figure 5, lower panel). Thus, it appears that DPA reacted, to an appreciable extent, with endogenous β-actin in intact RL34 cells. These results indicate that β-actin is an intracellular molecular target of Bio-DPA in RL34 cells. Modification of β-Actin by Catechol Type Polyphenols in RL34 Cells. Previously, Bo¨hl et al. reported that β-actin is a target protein for the flavonol quercetin (29). To examine the reactivity of the catechol type polyphenols, such as a flavonoid, to endogenous β-actin, RL34 cells were treated with 50 µM Bio-DPA after pretreatment with DPA and flavonoids (quercetin, EC, and ECG) for 1 h, and its modifications were analyzed by

Catechol Type Polyphenol May Modify Protein Sulfhydryls

Chem. Res. Toxicol., Vol. 22, No. 10, 2009 1695

purported to be the targets of electrophiles and ROS that, when modified, facilitate the derepression of Nrf2 and give rise to enzyme induction (19, 31). This leads us to assume that direct modification of Keap1 of DPA may regulate Keap1 and induce the gene expression. However, the modification of Keap1 by Bio-DPA was not detected by either the proteomic approach (Figure 4) or the pull-down assay (data not shown). These observations suggested that Keap1 is an additional target of DPA through another mechanism than covalent binding such as oxidation of intracellular sulfhydryls including GSH or protein cysteine residues.

Discussion

Figure 6. Modification of β-actin by catechol type polyphenols in RL34 cells. RL34 cells were pretreated with DPA and flavonoids (quercetin, EC, and ECG) for 1 h. The flavonoid-containing medium was removed by washing with PBS and replaced by the control medium containing 50 µM Bio-DPA for 1 h, and its modification was analyzed by Western blotting probed with HRP-avidin. (A) Pretreatment with DPA (0-500 µM) for 1 h. (B) Pretreatment with 50 µM flavonoids (quercetin, EC, and ECG) for 1 h. (C) Pretreatment with 50 µM vitamin C or vitamin E for 1 h.

Figure 7. Induction of the Keap1/Nrf2-regulated enzyme genes by DPA in RL34 cells. Incubation of RL34 cells with 0-100 µM DPA for 8 h. Dose-dependent effect of DPA on mRNA levels of NQO1 (upper), GSTP1 (middle), and GAPDH (lower).

Western blotting probed with HRP-avidin. As shown in Figure 6A, DPA (0-500 µM) pretreatment of RL34 cells resulted in a dose-dependent inhibition of the Bio-DPA modification. Furthermore, the formation of the Bio-DPA-modified β-actin was significantly decreased by pretreatment with the 50 µM flavonoids (Figure 6B). On the other hand, vitamin C or vitamin E pretreatments did not significantly inhibit the Bio-DPA modification (Figure 6C). This indicated that the antioxidant action of DPA or flavonoids does not contribute to the inhibition of the reactivity of Bio-DPA for β-actin. These results strongly suggest that β-actin is a potential target of the catechol type polyphenols in cellular proteins. Effect of DPA on Cytoprotective Enzyme Induction in RL34 Cells. It has been reported that dietary polyphenols have a potential to induce phase II drug-metabolizing enzyme expression (21, 30). As described above, electrophilic reaction of Bio-DPA to cellular proteins was inhibited by the flavonoid pretreatment (Figure 6B). Therefore, to investigate whether the electrophilicity of the catechol type polyphenol is involved in the cytoprotective enzyme induction, we examined the glutathione-S-transferase subunit-P1 (GSTP1)- and quinone oxidoreductase 1 (NQO1)-inducing ability of DPA. The incubation of RL34 cells with 0-100 µM DPA for 8 h led to a significant increase in the level of GSTP1 and NQO1 mRNA (Figure 7). Human Keap1 contains 27 cysteines, some of which are

Much attention has recently focused on the identification of a promising target protein that can interact with food chemicals. Quinones act as electrophiles and form covalent bonds with protein nucleophiles, irreversibly altering key cellular proteins, and individual quinones exhibit this reactivity to varying degrees (32). Dopamine quinone, which is an oxidation metabolite of dopamine, has been shown to covalently bind to reduced cysteine residues of proteins in vivo and in vitro (33, 34). Hence, we also note that quinone-mediated covalent binding through oxidation may also occur in other polyphenolic compounds. In this study, we demonstrated that the known reactions of catechol moieties with protein nucleophiles, such as thiol groups, can be exploited to study protein-polyphenol interactions and, furthermore, to screen for unknown protein targets of polyphenols of physiological and pharmacological interest. We have definitively indicated that the catechol type polyphenols, including Bio-DPA, have a high reactivity and selectivity for cysteine residues (Figures 1-3). The target identification was based on the proteomic approach (Figure 4) and pull-down technique (Figure 5). The inherent limitation of the technique lies in the required structural properties of polyphenol and protein that allow the use of chemical synthesis techniques. However, in the present experiments using Bio-DPA, we found that the approach may be undertaken successfully for a wide range of protein-polyphenol interactions (Figure 6). Further studies will address these problems, and an attempt will be made to identify all major target proteins of the catechol type polyphenols in human cells. Several lines of evidence indicate that ROS and electrophiles act as second messengers, representing an integral part of the cellular signal transduction network. The downstream effect of ROS and electrophiles is the oxidation of redox-sensible proteins through the direct modification of the thiol group of reactive cysteines (35, 36). Therefore, the modification of sulfhydryl groups in cellular proteins by catechol type polyphenols may cause the signal transductions through structural or functional alterations. We demonstrated here that β-actin is one of the major molecular targets of protein modification, not only by Bio-DPA (Figures 4 and 5) but also by flavonoids (Figure 6). Actin is one of the most abundant proteins in human tissues and serves essential functions as the cytoskeletal component in muscle and nonmuscle cells. Actin may exist as a monomer [globular actin (G-actin)] in the cell but readily polymerizes to form microfilaments [filamentous actin (F-actin)], rendering actin the major molecular player in the control of cell shape, cell adhesion, and cell motility (37). The dynamic reorganization of cellular actin is highly regulated, and ROS appear to be one vital regulatory element. Recent studies indicated that actin could constitute a direct target for oxidative modification (25, 38-41). Actin glutathionylation has already been reported in response to epidermal growth factor and in hepatocytes and hepatoma

1696

Chem. Res. Toxicol., Vol. 22, No. 10, 2009

cells exposed to oxidative stress (39, 40), and Cys272 and Cys374 were identified as reactive cysteines (42). Moreover, actin was shown to be oxidatively modified in pathophysiological states suggestive of oxidation as a cause of mechanical dysfunction (43). Thus, an explanation of the molecular basis of redox-regulated microfilament processes requires an understanding of the mechanism of assembly/disassembly and related changes of actin during redox conditions, as well as its regulation by associated proteins. Previously, actin cytoskeleton organization, that is, stress fibers, in adherent fibroblasts was changed upon tea catechin treatment (44). Furthermore, a microscopic analysis of quercetin targets in living cells revealed that F-actin rings, as found in Drosophila follicles, fluoresce brightly (45). In the light of these results, it seems likely that the flavonoids bind to actin in this special cytoplasmic structure, even though actin is apparently present in F-actin. This interpretation may also explain the observed quercetin-induced disruption of actin rings and of bone resorption in osteoclast-like cells (46). Although the authors speculated that quercetin might act on the signaling pathway involved in the assembly of actin rings, the present results may indicate that direct interaction of the catechol moiety with actin also modulates other signal transduction pathways. Induction of a family of cytoprotective enzyme genes encoding for proteins that protect against the damage of ROS and electrophiles is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many cytoprotective enzyme genes are regulated by upstream antioxidant response elements (AREs) that are targets of the leucine zipper transcription factor Nrf2 (19, 31). Under basal conditions, Nrf2 mainly resides in the cytoplasm bound to its cysteine-rich Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by oxidizing reactive cysteine residues of Keap1 (47, 48). The identification of β-actin as the molecular target of catechol type polyphenols led us to the assumption that actin scaffold proteins might also be involved in the polyphenol-induced modulation of the Keap1/Nrf-2 system. This assertion is based on the following observations: (i) Simple catechol compounds, as well as catechol type polyphenols, have been reported to induce cytoprotective enzyme expression and activity (21, 30), (ii) catechol type flavonoids bind to protein sulfhydryls, (iii) Keap1 binds to the actin cytoskeleton in the cytoplasm, and (iv) the modification of Keap1 by Bio-DPA was not detected by the present approach. Detection of modified Keap1 is difficult because the expression of Keap1 in cells is much lower than that of housekeeping proteins such as actin, one of the major targets of protein modification by catechol type polyphenols. In addition, the thiol-polyphenol adduct is reversible, which has also been reported for the GSH or GSTP1 adduct (49, 50). As a similar example, the Keap1 adduct of sulforaphane, one of the most famous electrophilic inducers of cytoprotective enzymes from cruciferous vegetables, has never been directly detected from the in vivo system because of its reversibility to hydrolysis and transacylation reactions (51). As another possibility, we propose the participation of the prooxidant actions of polyphenols. Most plant polyphenols possess both antioxidant as well as prooxidant properties (52). The cytotoxicities of quercetin were reported to be diminished by the supplement of H2O2 to the culture medium, while the addition of quercetin was found to attenuate the H2O2-induced cytotoxicity (53). Furthermore, in vitro studies indicated that tea catechins are unstable in several cell culture media (14, 15). The oxidation of polyphenols and the resulting production of ROS have been

Ishii et al.

considered responsible for biological activities such as receptor inactivation and telomerase inhibition (54, 55). Conversely, it has been suggested that although free quinones are short-lived in vivo (56), the binding of quinones to a protein may dramatically extend the half-life of these reactive species (56, 57). Furthermore, the intracellular ROS production by cysteine- and protein-bound polyphenols was reported (33, 58). Therefore, modification of the intracellular actin by catechol type polyphenols may induce the oxidation of GSH and other protein sulfhydryls, such as Keap1, through prooxidant actions. In fact, down-regulation of the intracellular GSH level enhanced the cytoprotective enzyme induction by flavonoids, supporting the prooxidant theory (59). This finding is in line with other literature reports demonstrating that the prooxidant action of catechol type polyphenols rather than their antioxidant activity is important for their health-promoting property (59, 60). Future studies need to clarify this point and address the question of whether the catechol group is able to inhibit the differential functions of actin in the nucleus and cytoplasm, such as with the Keap1/Nrf-2 system. In conclusion, this study indicates that β-actin is one of the major targets of protein modification by catechol type polyphenols and provides an alternative approach to understand that catechol type polyphenol is a potential modifier of redoxdependent cellular events through sulfhydryl modification. The results also encourage further investigation into the biological activities of polyphenols. In addition, the present results may not reflect bindings of dietary polyphenols in vivo entirely, because a biotinylation of DPA may hinder the ability of DPA to access protein cysteine residues. It is still uncertain and needed to elucidate that catechol type polyphenol first targets β-actin in cultured cells and that these bindings are associated with its bioactivities. Searching for high affinity proteins that bind to the polyphenolic flavonoid is the first step to understanding the molecular and biochemical mechanisms of the functional effects of dietary polyphenols. Our results provide a foundation for future studies about the mechanism of action of dietary polyphenols.

References (1) Bravo, L. (1998) Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutr. ReV. 56, 317–333. (2) Scalbert, A., Manach, C., Morand, C., Re´me´sy, C., and Jime´nez, L. (2005) Dietary polyphenols and the prevention of diseases. Crit. ReV. Food Sci. Nutr. 45, 287–306. (3) Stadtman, E. R. (2001) Protein oxidation in aging and age-related diseases. Ann. N.Y. Acad. Sci. 928, 22–38. (4) Arts, I. C., and Hollman, P. C. (2005) Polyphenols and disease risk in epidemiologic studies. Am. J. Clin. Nutr. 81 (I1 Suppl.), 317S– 325S. (5) Dragsted, L. O. (2003) Antioxidant actions of polyphenols in humans. Int. J. Vitam. Nutr. Res. 73, 112–119. (6) Galati, G., and O’Brien, P. J. (2003) Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radical Biol. Med. 37, 287–303. (7) Kampa, M., Nifli, A. P., Notas, G., and Castanas, E. (2007) Polyphenols and cancer cell growth. ReV. Physiol. Biochem. Pharmacol. 159, 79–113. (8) Sazuka, M., Itoi, T., Suzuki, Y., Odani, S., Koide, T., and Isemura, M. (1996) Evidence for the interaction between (-)-epigallocatechin gallate and human plasma proteins fibronectin, fibrinogen, and histidine-rich glycoprotein. Biosci., Biotechnol., Biochem. 60, 1317– 1319. (9) Wang, X., Song, K. S., Guo, Q. X., and Tian, W. X. (2003) Green tea (-)-epigallocatechin gallate can inhibit FAS in Vitro. Biochem. Pharmacol. 66, 2039–2047. (10) Dufour, C., and Dangles, O. (2005) Flavonoid-serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy. Biochim. Biophys. Acta 1721, 164–173. (11) Bae, M. J., Ishii, T., Minoda, K., Kawada, Y., Ichikawa, T., Mori, T., Kamihira, M., and Nakayama, T. (2009) Albumin stabilizes (-)-

Catechol Type Polyphenol May Modify Protein Sulfhydryls

(12) (13)

(14)

(15)

(16)

(17)

(18)

(19)

(20) (21) (22)

(23)

(24)

(25) (26)

(27)

(28)

(29)

(30) (31)

epigallocatechin gallate in human serum: Binding capacity and antioxidant property. Mol. Nutr. Food Res. 53, 709–715. Tachibana, H., Koga, K., Fujimura, Y., and Yamada, K. (2004) A receptor for green tea polyphenol EGCG. Nat. Struct. Mol. Biol. 11, 380–381. Ermakova, S., Choi, B. Y., Choi, H. S., Kang, B. S., Bode, A. M., and Dong, Z. (2005) The intermediate filament protein vimentin is a new target for epigallocatechin gallate. J. Biol. Chem. 280, 16882– 16890. Nakayama, T., Ichiba, M., Kuwabara, M., Kajiya, K., and Kumazawa, S. (2002) Mechanisms and structural specificity of hydrogen peroxide formation during oxidation of catechins. Food Sci. Technol. Res. 8, 261–267. Long, L. H., Clement, M. V., and Halliwell, B. (2000) Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem. Biophys. Res. Commun. 25, 50–53. Awad, H. M., Boersma, M. G., Boeren, S., Van Bladeren, P. J., Vervoort, J., and Rietjens, I. M. (2003) Quenching of quercetin quinone/quinone methides by different thiolate scavengers: stability and reversibility of conjugate formation. Chem. Res. Toxicol. 16, 822– 831. Galati, G., Moridani, M. Y., Chan, T. S., and O’Brien, P. J. (2001) Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation. Free Radical Biol. Med. 30, 370–382. Ishii, T., Mori, T., Tanaka, T., Mizuno, D., Yamaji, R., Kumazawa, S., Nakayama, T., and Akagawa, M. (2008) Covalent modification of proteins by green tea polyphenol (-)-epigallocatechin-3-gallate through autoxidation. Free Radical Biol. Med. 45, 1384–1394. Wakabayashi, N., Dinkova-Kostova, A. T., Holtzclaw, W. D., Kang, M. I., Kobayashi, A., Yamamoto, M., Kensler, T. W., and Talalay, P. (2004) Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization. Proc. Natl. Acad. Sci. U.S.A. 101, 2040–2045. Liu, B., Chen, Y., and St Clair, D. K. (2008) ROS and p53: A versatile partnership. Free Radical Biol. Med. 44, 1529–1535. Na, H. K., and Surh, Y. J. (2006) Transcriptional regulation via cysteine thiol modification: A novel molecular strategy for chemoprevention and cytoprotection. Mol. Carcinog. 45, 368–380. Nakamura, Y., Torikai, K., Ohto, Y., Murakami, A., Tanaka, T., and Ohigashi, H. (2000) A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose- and timing-dependent enhancement and involvement of bioactivation by tyrosinase. Carcinogenesis 21, 1899–1907. Shibata, T., Yamada, T., Ishii, T., Kumazawa, S., Nakamura, H., Masutani, H., Yodoi, J., and Uchida, K. (2003) Thioredoxin as a molecular target of cyclopentenone prostaglandins. J. Biol. Chem. 278, 26046–26054. Ishii, T., Tatsuda, E., Kumazawa, S., Nakayama, T., and Uchida, K. (2003) Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: Identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 42, 3474– 3480. Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y., and Liebler, D. C. (2008) Cytosolic and nuclear protein targets of thiol-reactive electrophiles. Chem. Res. Toxicol. 19, 20–29. Nakamura, Y., Torikai, K., and Ohigashi, H. (2001) A catechol antioxidant protocatechuic acid potentiates inflammatory leukocytederived oxidative stress in mouse skin via a tyrosinase bioactivation pathway. Free Radical Biol. Med. 30, 967–978. Loecken, E. M., and Guengerich, F. P. (2008) Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with biselectrophiles produce DNA-protein cross-links but not mutations. Chem. Res. Toxicol. 21, 453–458. Bolton, J. L., Turnipseed, S. B., and Thompson, J. A. (1997) Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: Studies utilizing amino acid and peptide models. Chem.-Biol. Interact. 107, 185–200. Bo¨hl, M., Czupalla, C., Tokalov, S. V., Hoflack, B., and Gutzeit, H. O. (2005) Identification of actin as quercetin-binding protein: An approach to identify target molecules for specific ligands. Anal. Biochem. 346, 295–299. Na, H. K., and Surh, Y. J. (2008) Modulation of Nrf2-mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG. Food Chem. Toxicol. 46, 1271–1278. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., O’Connor, T., and Yamamoto, M. (2003) Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells 8, 379–391.

Chem. Res. Toxicol., Vol. 22, No. 10, 2009 1697 (32) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinines in toxicology. Chem. Res. Toxicol. 13, 135–160. (33) Akagawa, M., Ishii, Y., Ishii, T., Shibata, T., Yotsu-Yamashita, M., Suyama, K., and Uchida, K. (2006) Metal-catalyzed oxidation of protein-bound dopamine. Biochemistry 45, 15120–15128. (34) LaVoie, M. J., Ostaszewski, B. L., Weihofen, A., Schlossmacher, M. G., and Selkoe, D. J. (2005) Dopamine covalently modifies and functionally inactivates parkin. Nat. Med. 11, 1214–1221. (35) Rhee, S. G. (2006) Cell signaling. H2O2, a necessary evil for cell signaling. Science 312, 1882–1883. (36) Na, H. K., and Surh, Y. J. (2006) Transcriptional regulation via cysteine thiol modification: A novel molecular strategy for chemoprevention and cytoprotection. Science 45, 368–380. (37) Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. ReV. Biophys. Biomol. Struct. 29, 545–576. (38) Fiaschi, T., Cozzi, G., Raugei, G., Formigli, L., Ramponi, G., and Chiarugi, P. (2006) Redox regulation of β-actin during integrinmediated cell adhesion. J. Biol. Chem. 281, 22983–22991. (39) 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. (40) Lind, C., Gerdes, R., Hamnell, Y., Schuppe-Koistinen, I., von Lo¨wenhielm, H. B., Holmgren, A., and Cotgreave, I. A. (2002) Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch. Biochem. Biophys. 99, 229–240. (41) Ishii, T., and Uchida, K. (2004) Induction of reversible cysteinetargeted protein oxidation by an endogenous electrophile 15-deoxy∆12,14-prostaglandin J2. Chem. Res. Toxicol. 17, 1313–1322. (42) Lassing, I., Schmitzberger, F., Bjo¨rnstedt, M., Holmgren, A., Nordlund, P., Schutt, C. E., and Lindberg, U. (2007) Molecular and structural basis for redox regulation of β-actin. J. Mol. Biol. 370, 331–348. (43) Pastore, A., Tozzi, G., Gaeta, L. M., Bertini, E., Serafini, V., Di Cesare, S., Bonetto, V., Casoni, F., Carrozzo, R., Federici, G., and Piemonte, F. (2003) Actin glutathionylation increases in fibroblasts of patients with Friedreich’s ataxia: A potential role in the pathogenesis of the disease. J. Biol. Chem. 278, 42588–42595. (44) Hung, C. F., Huang, T. F., Chiang, H. S., and Wu, W. B. (2005) (-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms. J. Cell Biochem. 96, 183–197. (45) Gutzeit, H. O., Henker, Y., Kind, B., and Franz, A. (2004) Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence. Biochem. Biophys. Res. Commun. 318, 490–495. (46) Woo, J. T., Nakagwa, H., Notoya, M., Yonezawa, T., Udagawa, N., Lee, I. S., Ohnishi, M., Hagiwara, H., and Nagai, K. (2004) Quercetin suppresses bone resorption by inhibiting the differentiation and activation of osteoclasts. Biol. Pharm. Bull. 27, 504–509. (47) Eggler, A. L., Liu, G., Pezzuto, J. M., van Breemen, R. B., and Mesecar, A. D. (2005) Modifying specific cysteines of the electrophilesensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2. Proc. Natl. Acad. Sci. U.S.A. 102, 10070–10075. (48) Eggler, A. L., Luo, Y., van Breemen, R. B., and Mesecar, A. D. (2007) Identification of the highly reactive cysteine 151 in the chemopreventive agent-sensor Keap1 protein is method-dependent. Chem. Res. Toxicol. 20, 1878–1884. (49) Boersma, M. G., Vervoort, J., Szymusiak, H., Lemanska, K., Tyrakowska, B., Cenas, N., Segura-Aguilar, J., and Rietjens, I. M. (2000) Regioselectivity and reversibility of the glutathione conjugation of quercetin quinone methide. Chem. Res. Toxicol. 13, 185–191. (50) van Zanden, J. J., Ben Hamman, O., van Iersel, M. L., Boeren, S., Cnubben, N. H., Lo Bello, M., Vervoort, J., van Bladeren, P. J., and Rietjens, I. M. (2003) Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin. Chem.-Biol. Interact. 145, 139–148. (51) Hong, F., Freeman, M. L., and Liebler, D. C. (2005) Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane. Chem. Res. Toxicol. 18, 1917–1926. (52) Sergediene, E., Jo¨nsson, K., Szymusiak, H., Tyrakowska, B., Rietjens, I. M., and Cenas, N. (1999) Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: Description of quantitative structureactivity relationships. FEBS lett. 462, 392–396. (53) Robaszkiewicz, A., Balcerczyk, A., and Bartosz, G. (2007) Antioxidative and prooxidative effects of quercetin on A549 cells. Cell Biol. Int. 31, 1245–1250. (54) Hou, Z., Sang, S., You, H., Lee, M. J., Hong, J., Chin, K. V., and Yang, C. S. (2005) Mechanism of action of (-)-epigallocatechin3-gallate: auto-oxidation-dependent inactivation of epidermal growth

1698

Chem. Res. Toxicol., Vol. 22, No. 10, 2009

factor receptor and direct effects on growth inhibition in human esophageal cancer KYSE 150 cells. Cancer Res. 65, 80498056. (55) Naasani, I., Oh-Hashi, F., Oh-Hara, T., Feng, W. Y., Johnston, J., Chan, K., and Tsuruo, T. (2003) Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in Vitro and in ViVo. Cancer Res. 63, 824830. (56) Graham, D. G. (1978) Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharmacol. 14, 633–643. (57) Paz, M. A., Fluchiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) Specific detection of quinoproteins by redox-cycling staining. J. Biol. Chem. 266, 689–692.

Ishii et al. (58) Izumi, Y., Sawada, H., Yamamoto, N., Kume, T., Katsuki, H., Shimohama, S., and Akaike, A. (2005) Iron accelerates the conversion of dopamine-oxidized intermediates into melanin and provides protection in SH-SY5Y cells. J. Neurosci. Res. 82, 126–137. (59) Lee-Hilz, Y. Y., Boerboom, A. M., Westphal, A. H., Berkel, W. J., Aarts, J. M., and Rietjens, I. M. (2006) Pro-oxidant activity of flavonoids induces EpRE-mediated gene expression. Chem. Res. Toxicol. 19, 1499–1505. (60) Azam, S., Hadi, N., Khan, N. U., and Hadi, S. M. (2004) Prooxidant property of green tea polyphenols epicatechin and epigallocatechin3-gallate: Implications for anticancer properties. Toxicol. in Vitro 18, 555–561.

TX900148K