From the
Laboratory of Food and Biodynamics,
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya
464-8601, Japan,
Department of Food and
Nutritional Science, University of Shizuoka, Shizuoka 422-8529, Japan, and
¶Department of Biological Responses, Institute
for Virus Research, Kyoto University, Kyoto 606-8507, Japan
Received for publication, April 9, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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Thioredoxin 1 (Trx) is a small and ubiquitously expressed protein originally identified in Escherichia coli and is evolutionarily conserved from prokaryotes to higher eukaryotes (1719). Human thioredoxin was cloned as an adult T cell leukemia-derived factor or interleukin-1-like factor (20, 21). Trx is an essential cofactor electron donor for ribonucleotide reductase but also has many other cellular functions, including regulation of transcription factors and apoptosis, and can act exogenously as a redox active growth factor (18, 22). In addition, Trx is known to play important roles in the redox regulation of signal transduction and in cytoprotection against oxidative stress (23, 24). The catalytic activity of Trx resides in its active site where the two redox-active cysteine residues (Cys-32 and Cys-35) undergo reversible oxidation/reduction. In addition to the conserved cysteine residues in the active site, three additional structural cysteine residues (Cys-62, Cys-69, and Cys-73) are present in the structure of the human Trx. The mechanisms for the reducing action of Trx is that substrate (X-S2) binds to a conserved hydrophobic surface and, in the hydrophobic environment of the complex, the thiolate of Cys-32, acting as a nucleophile, combines with the protein substrate to form a covalently linked mixed disulfide (-Cys-32-S-S-X). Finally, the deprotonated Cys-35 attacks the -Cys-32-S-S-X disulfide bond, releasing the reduced protein substrate and forming Trx-Cys-32-Cys-35-disulfide, which is then reduced by Trx reductase (25).
In our previous study, based on an extensive screening of diverse chemical
agents on the induction of intracellular production of reactive oxygen species
(ROS), we identified cyclopentenone PGs, such as 15d-PGJ2, as the
potential inducers of intracellular oxidative stress in SH-SY5Y human
neuroblastoma cells (14). As
the intracellular events associated with the PG-induced oxidative stress, we
observed (i) the cellular redox alteration represented by depletion of
antioxidant defenses, such as glutathione and glutathione peroxidase, (ii) a
transient decrease in the mitochondrial membrane potential (),
(iii) the production of protein-bound lipid peroxidation products, such as
acrolein and 4-hydroxy-2-nonenal, and (iv) the accumulation of ubiquitinated
proteins. In addition, the thiol compound, N-acetylcysteine,
significantly inhibited the PG-induced ROS production, thereby preventing
cytotoxicity. These observations suggested that the redox alteration was
closely related to the pro-oxidant effect of the cyclopentenone PGs. In the
present study, to investigate the correlation between the redox regulation and
15d-PGJ2-induced oxidative stress and to establish the cellular
mechanism for protection against the endogenous electrophile, we investigated
the role of Trx in 15d-PGJ2-induced oxidative cell injury.
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EXPERIMENTAL PROCEDURES |
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Cell CultureSH-SY5Y cells were grown in Cosmedium-001 (CosmoBio, Tokyo, Japan) containing 5% Nakashibethu precolostrum newborn calf serum, 100 µg/ml penicillin, and 100 units/ml streptomycin. The cells were seeded in plates coated with poly-lysine and cultured at 37 °C. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (14).
Flow Cytometry Analysis of Intracellular ROS Production DCFH-DA was used to measure the ROS (27, 28). Cells were incubated with 10 µM 2',7'-dichlorofluorescein diacetate (dissolved in Me2SO) for 30 min at 37 °C and then treated with different agents for an additional 30 min at 37 °C. After chilling on ice, the cells were washed with ice-cold PBS, scraped from the plate, and resuspended at 1 x 106 cells/ml in PBS containing 10 mM EDTA. The fluorescence was measured using a flow cytometer (Epics XL, Beckman Coulter).
Preparation of Biotinylated 15d-PGJ2The carboxyl group of 15d-PGJ2 was modified by amidation with EZ-link 5-(biotinamido)pentylamine by a modification of a previously described procedure (16). Biotinylated 15d-PGJ2 was purified through a reverse-phase HPLC eluted with a linear gradient of acetonitrile/water/acetic acid. The modified PG was then dried under argon and dissolved in Me2SO for further use.
Immunochemical Detection of Biotinylated 15d-PGJ2-binding ProteinCells 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 2% bovine serum albumin and the primary anti-biotin antibody. The cells were then incubated for 1 h in the presence of fluorescein isothiocyanate-labeled goat anti-mouse IgG (Amersham Biosciences), rinsed with PBS containing 0.3% Triton X-100, and covered with anti-fade solution. Images of the cellular immunofluorescence were acquired using a confocal laser scanning microscope (Fluoroview; Olympus Optical Co., Ltd., Tokyo, Japan) with a x40 objective (488-nm excitation and 518-nm emission).
SDS-PAGE and Immunoblot AnalysisSDS-PAGE was performed according to Laemmli (29). A gel was transblotted onto a nitrocellulose membrane, incubated with Block Ace (40 mg/ml) for blocking, and washed with TBS containing 0.05% Tween 20 (TBS/Tween). This procedure was followed by the addition of HRP-conjugated NeutrAvidin or anti-mouse IgG antibody and ECL reagents (Amersham Biosciences). The bands were visualized by Cool Saver AE-6955 (ATTO, Tokyo, Japan).
Stable Transfection with Trx in SH-SY5Y CellsSH-SY5Y cells
were transfected with pcTrx-1 vector or with the vector alone using
GenePORTERTM transfection reagent (Gene Therapy Systems, Inc.). In these
experiments, 1 x 106 cells were incubated with
DNA-GenePORTERTM mixture (2 µg of DNA/10 µl of GenePORTERTM)
in 1 ml of serum-free Opti-MEM (Invitrogen) at 37 °C. After 6 h of
incubation, 1 ml of complete medium was added, and cells were cultured for 18
h. Thereafter, stable transfectants were isolated by selection on 500 µg/ml
G418 for 3 weeks. Single clones of the stably transfected cells were
isolated by limiting dilution. Several G418-resistant stable clones were
maintained in medium containing 500 µg/ml G418.
Biotinylated 15d-PGJ2 Labeling of Trx in SH-SY5Y CellsSH-SY5Y cells at 50% confluence were incubated with 50 µM biotinylated 15d-PGJ2 for 1 h. The cells were washed with PBS, harvested, and lysed in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1% SDS, 1% Triton, plus protease inhibitors. Cell lysates containing 200 µg of protein were incubated batch-wise with 50 µl of NeutrAvidin-Plus beads overnight at 4 °C with constant shaking. The beads were rinsed three times with lysis buffer by centrifugation at 6,000 rpm for 1 min. The proteins were eluted by boiling the beads in Laemmli sample buffer for 5 min and analyzed by SDS-PAGE followed by immunodetection with anti-Trx monoclonal antibody. In addition, the cell lysates were incubated with 3 µg of anti-Trx antibody overnight at 4 °C. The mixture was then treated with 50 µl of protein G-Sepharose (Amersham Biosciences) and incubated for 1 h at 4 °C. The mixture was then centrifuged (2,000 rpm, 5 min), rinsed three times with lysis buffer, and then boiled with the Laemmli sample buffer, and the biotinylated proteins were then subjected to immunoblot and detection with HRP-conjugated NeutrAvidin and ECL.
Enzyme-linked Immunosorbent Assay (ELISA)To coat the wells of the microtiter plate, 100 µl/well hrTrx (0.1 mg/ml) in PBS was used and then incubated overnight at 4 °C. Following washing with TBS/Tween, 100 µl of the biotinylated 15d-PGJ2 solution was added to the wells. After incubation for 2 h at 37 °C followed by washing with TBS/Tween, each well was filled with 200 µl of Block Ace solution (4 mg/ml) for 1 h at 37 °C. The HRP-conjugated NeutrAvidin was then added to the wells at 100 µl/well for 1 h at 37 °C. After washing, 100 µl of 0.05 M citrate buffer, pH 5.0, containing 0.4 mg/ml o-phenylenediamine and 0.003% H2O2 was added and incubated for several minutes at room temperature in the dark. The reaction was terminated by the addition of 2 M sulfuric acid, and the absorbance at 492 nm was read on a micro-ELISA plate reader.
Titration of Trx Sulfhydryls with DTNBThe reactivity of the sulfhydryl groups in hrTrx was determined by titration with DTNB by the method of Ellman (30), as modified by Riddles et al. (31). A 0.1 mg/ml hrTrx sample was treated with 0.1 mM 15d-PGJ2, and after dialysis against PBS, the protein was denatured with 8 M guanidine hydrochloride containing 13 mM EDTA and 133 mM Tris, pH 8, and reacted with DTNB (1 mM) at room temperature for 5 min. The reaction was monitored as an increase in absorbance at 412 nm. The concentration of sulfhydryl groups was calculated using a standard curve with N-acetylcysteine.
Matrix-assisted Laser Desorption Ionization Time-of-flight Mass
Spectrometry (MALDI-TOF MS)The native and
15d-PGJ2-modified hrTrx were mixed with a saturated solution of
3,5-dimethoxy-4-hydroxycinnamic acid (Sigma) containing 75% acetonitrile and
0.1% trifluoroacetic acid and dried on stainless steel targets at room
temperature and pressure. The trypsin-digested hrTrx were mixed with a
saturated solution of -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 an
Autoflex matrix-assisted laser desorption ionization time-of-flight mass
spectrometer (Bruker, Bremen, Germany) with a nitrogen laser (337 nm). All
analyses were in the positive ion mode, and the instrument was calibrated
immediately prior to each series of studies.
Electrospray Ionization-Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (ESI-LC/MS/MS) AnalysisThe ESI-LC/MS (and ESI-LC/MS/MS) analyses were performed on an LCQ ion trap mass system (ThermoQuest) equipped with an electrospray ion source. The electrospray system employed a 5-kV spray voltage and a capillary temperature of 260 °C.
Peptide MappingThe native and 15d-PGJ2-modified hrTrx (1 mg/ml) were digested with modified trypsin in 0.25 ml 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). Peptide samples were analyzed by a reversed-phase HPLC, a system that consisted of a nanospace SI-1 HPLC system (SHISEIDO Co., Ltd., Tokyo, Japan) with a FP-1520 fluorescence detector (Jasco Co., Tokyo, Japan), using a Capcell Pak C18 UG120 column (2.0 x 250-mm inner diameter; SHISEIDO Co., Ltd., Tokyo, Japan). These samples were eluted with a linear gradient of water containing 0.1% formic acid (Solvent A) and acetonitrile containing 0.08% formic acid (Solvent B) (time = 03 min, 10% B; 345 min, 1040% B; 4550 min, 4050% B; 5052 min, 5080% B). The flow rate was 0.2 ml/min, and the column temperature was controlled at 40 °C. The chromatograms were recorded at 215 nm.
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RESULTS |
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Effect of Trx Overexpression on 15d-PGJ2-induced Oxidative StressThe fact that cyclopentenone PGs are susceptible to nucleophilic addition reactions with thiols suggests that the action of the cyclopentenone PGs are closely related to the direct reaction with glutathione and/or other thiol compounds. However, L-buthionine-S,R-sulfoximine, a specific inhibitor of glutathione biosynthesis, itself, did not so effectively induce intracellular ROS production and cell death, indicating that the effects of the cyclopentenone PGs may not merely result from glutathione depletion alone (14). This and the finding that the electrophilic center of 15d-PGJ2 is involved in the prooxidant effect (Fig. 1) suggested that other cellular redox molecules might play crucial roles in protection against 15d-PGJ2-induced oxidative cell injury. Based on the previous findings that Trx, a key molecule in the maintenance of cellular redox balance, plays critical roles in protecting against oxidative stress and mediating signal transduction (35), we investigated the role of this redox regulator on the 15d-PGJ2-induced ROS production. To this end, we established the Trx-overexpressing derivatives of SH-SY5Y cells by stable transfection with Trx cDNA. The Trx expression vector (pcTrx-1) was introduced into the SH-SY5Y cells with the selectable pcDNA, and four clones with resistance to G418 were selected. As a control, SH-SY5Y cells transfected with a control vector were similarly selected for G418 resistance. Four clones were isolated. As shown in Fig. 2A, the Trx-transfected clones Trx-1 and Trx-3 demonstrated a readily detectable expression of the Trx protein by immunoblot analysis with the anti-Trx monoclonal antibody, whereas the control transfected cells Neo-1, -2, -3, and -4 did not.
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For the experiments shown below, the results were compared between Neo-2 and Trx-3, to assess the effectiveness of expressed Trx in protection against 15d-PGJ2-induced cell injury. First, we measured the intracellular ROS production by flow cytometry analysis. As shown in Fig. 2B, the ROS production in the control SH-SY5Y was significantly induced by 15d-PGJ2. The level of ROS in the cells exposed to 15d-PGJ2 (50 µM) was about 5-fold higher than that of the vehicle-treated cells, whereas the intracellular ROS production in the Trx-overexpressed cells exposed to 15d-PGJ2 was inhibited about 40% of the vector control. Next, we examined the effect of Trx on the 15d-PGJ2-induced cytotoxicity. The transfected SH-SY5Y cells were examined by MTT assay for sensitivity to cytotoxicity induced by continuous exposure (24 h) to 15d-PGJ2. As shown in Fig. 2C, 15d-PGJ2 (5 or 10 µM) resulted in a decrease in the MTT reduction levels to 10% of the basal levels on vector control cells within 24 h of exposure, whereas the Trx-overexpressing cells were more resistant to 15d-PGJ2-induced cell death than the control cells. We also examined the effect of Trx overexpression on the accumulation of ubiquitinated proteins and p53, both of which have been suggested to be involved in the 15d-PGJ2-induced cell death (14, 15), and found that Trx could have significantly inhibited the accumulation of these proteins (data not shown). These data strongly suggest that Trx may be involved, at least in part, in the protection against the 15d-PGJ2 cytotoxicity.
Covalent Binding of 15d-PGJ2 to Endogenous
Trx in SH-SY5Y CellsBecause Trx contains reactive sulfhydryl
groups, it can be hypothesized that Trx may directly react with
15d-PGJ2. Apart from low molecular weight
15d-PGJ2-glutathione adduct, the 15d-PGJ2-thiol
conjugates are associated with high molecular weight proteins. The list of
15d-PGJ2-modified proteins so far includes IB kinase
(10), Trx reductase
(36), and
keap1.2 Accordingly,
we studied the direct interaction between 15d-PGJ2 and endogenous
Trx in SH-SY5Y cells. To determine whether 15d-PGJ2 reacts with Trx
or other proteins, we prepared a biotinylated 15d-PGJ2
(Fig. 3A), which
retains the
,
-unsaturated ketone substituent and the
electrophilic
-carbon of 15d-PGJ2. As shown in
Fig. 3B, incorporation
of the biotinylated 15d-PGJ2 into the cells was observed in SH-SY5Y
cells by immunocytochemical detection, suggesting the utility of the
biotinylated 15d-PGJ2. We then attempted to detect the
15d-PGJ2-Trx adduct in the cells exposed to the biotinylated
15d-PGJ2. To this end, the SH-SY5Y cells were treated with 50
µM biotinylated 15d-PGJ2 for 1 h, and the cell lysate
was incubated with NeutrAvidin beads. After washing with lysis buffer,
proteins bound to the resin through biotinylated 15d-PGJ2 were
eluted with SDS-PAGE sample buffer, and Trx was detected by immunoblot
analysis with the anti-Trx monoclonal antibody
(Fig. 3C).
Alternatively, cell lysates were subjected to immunoprecipitation with an
anti-Trx monoclonal antibody, and the presence of biotinylated
15d-PGJ2-modified proteins was detected by immunoblot analysis with
HRP-conjugated NeutrAvidin (Fig.
3C). Thus, it appeared that 15d-PGJ2 reacted
to an appreciable extent with endogenous Trx, itself, in intact SH-SY5Y
cells.
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Covalent Binding of 15d-PGJ2 to Human Recombinant Trx Covalent binding of 15d-PGJ2 to Trx also occurred upon in vitro incubation of hrTrx with biotinylated 15d-PGJ2. As shown in Fig. 4A, the biotinylated 15d-PGJ2 was bound to hrTrx in a dose-dependent manner. In addition, binding of the biotinylated 15d-PGJ2 to hrTrx was also shown by ELISA analysis (Fig. 4B). Moreover, free sulfhydryl groups within the 15d-PGJ2-treated hrTrx were titrated with DTNB. As shown in Fig. 4C, the exposure of hrTrx to 15d-PGJ2 resulted in the loss of cysteine residues, and approximately two cysteine residues per hrTrx were lost after 1 h; however, 9,10-dihydro-15d-PGJ2 did not. These data suggest that 15d-PGJ2 modifies the free sulfhydryl groups within the hrTrx molecules.
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MALDI-TOF MS Analysis of 15d-PGJ2-treated hrTrxTo further elucidate the mechanism for modification of Trx by 15d-PGJ2, we attempted to identify the modification sites of the protein by monitoring the formation of the 15d-PGJ2 Michael adducts by mass spectrometric methods. As shown in Fig. 5, the MALDI-TOF MS analysis of the native hrTrx revealed a peak of m/z 11,785. When hrTrx was incubated with 0.1 mM 15d-PGJ2 in 50 mM sodium phosphate buffer, pH 7.4, for 2 h at 37 °C, some unmodified hrTrx subunits were observed, as was the peak (m/z 12,099) corresponding to the addition of one molecule of 15d-PGJ2 per protein. Further incubations resulted in the appearance of peaks corresponding to the addition of one to two molecules of 15d-PGJ2 (data not shown).
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Identification of the 15d-PGJ2 Modification Site in hrTrxTo confirm the 15d-PGJ2 modification sites, the native and 15d-PGJ2-treated hrTrx were digested with trypsin and then analyzed by ESI-LC/MS. Peptide mass mapping by ESI-LC/MS analysis of the tryptic peptides from the native hrTrx provided identification of the peptides accounting for 92% of the protein sequence (see Fig. 6A and Table I). Relative to the calculated mass of the unmodified peptide, which showed an increased mass of +316 Da corresponding to the addition of a single molecule of 15d-PGJ2, namely Tp-1 and Tp-2, were detected by ESI-LC/MS analysis (Fig. 6A). Moreover, these two peptides were also detected in the MALDI-TOF MS analysis of tryptic peptides from the 15d-PGJ2-treated hrTrx (see Fig. 6B and Table I).
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To identify the 15d-PGJ2-modification site, the 15d-PGJ2-modified peptides, Tp-1 and Tp-2, were further analyzed by ESI-LC/MS/MS without additional chromatography. The MS/MS spectrum of the [M + H]+ at m/z 1,940.7 from the 15d-PGJ2-modified peptide (Tp-1; LVVVDFSATWCGPCK) is shown in Fig. 7. In the MS/MS analysis, the singly charged N-terminal product ions (b8 and b11) and its H2O loss ions (b618, b918, b1018, and b1218) were observed. The C-terminal fragment ions (y4, y5, y6, y7, y8, y9, y10, y11, y12, and y13), N-terminal product ion (b14), and the H2O loss fragment ion (y318) were observed to increase 316 Da, suggesting that the 15d-PGJ2 modification site is in the sequence on Cys-35 but not Cys-32. The MS/MS spectrum of the [M + 2H]2+ at m/z 1,518.9 from the 15d-PGJ2-modified peptide (Tp-2; YSNVIFLEVDVDDCQDVASECEVK) is shown in Fig. 8. In the MS/MS analysis, the singly charged N-terminal product ions (b7, b8, b9, b11, b12, b13, b15, b16, and b17) and its H2O loss fragment ions (b618, b718, b818, b918, b1118, b1418, b1518, b1618, and b1718) and doubly charged N-terminal product ions (b202+ and b212+) were observed. The singly charged C-terminal product ions (y4, y5, y6, y8, y9, y11, y12, y13, y14, and y15) and doubly charged product ions (y182+, y192+, and b232+) were observed to increase 316 Da. These data suggest that the 15d-PGJ2 modification is associated with Cys-69 on the peptide Tp-2. On the other hand, the modification of the other cysteine residues, Cys-62 and Cys-73, were not detected (data not shown). Thus, we identified the two target cysteine residues, Cys-35 and Cys-69, of the 15d-PGJ2 modification in Trx.
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DISCUSSION |
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In our previous study (14),
as part of an effort to identify the endogenous inducer of intracellular
oxidative stress and to elucidate the molecular mechanism underlying the
oxidative stress-mediated cell degeneration, we examined the oxidized fatty
acid metabolites for their ability to induce intracellular ROS production in
SH-SY5Y cell in vitro and found that the J2 series of the
PGs represents the most potent inducers. This and the observations that the
intracellular ROS production was accompanied by the alteration of the cellular
redox status and the production of lipid peroxidation-derived highly cytotoxic
aldehydes, such as acrolein and 4-hydroxy-2-nonenal, which could also induced
the intracellular ROS production, suggested that intracellular oxidative
stress constitutes a pivotal step in the pathway of cellular dysfunction
induced by the PGs. In the present study, to further establish the molecular
mechanism underlying the PG-induced cell injury, we examined the effect of
9,10-dihydro-15d-PGJ2, an analog of 15d-PGJ2, on the
induction of ROS production and cell damage and found that the reduction of
the double bond in the cyclopentenone ring of 15d-PGJ2 virtually
abolished the pro-oxidant and cytotoxic effects of 15d-PGJ2
(Fig. 1). Thus, the pro-oxidant
action of 15d-PGJ2 appeared to operate via mechanisms that depend
upon the reactivity of its electrophilic ,
-unsaturated
ketones.
The reactive center of the cyclopentenone PGs has been proposed to account for some of their receptor-independent biological actions (10, 51). They can covalently react by means of the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins that play an important role in the control of the redox cell-signaling pathways (10, 51, 52). This, in turn, suggested that cellular redox molecules might play crucial roles in the regulation of the biological functions of cyclopentenone PGs. In the present study, we investigated the role of Trx on the 15d-PGJ2-induced oxidative cell damage. Trx, a key molecule in the maintenance of the cellular redox balance, has been shown to play critical roles in protecting against oxidative stress and mediating signal transduction (53). Indeed, the overexpression of cytoplasmic Trx protected against oxidative stress-induced cell death (35, 54). Andoh et al. (55) have also shown that, using serum deprivation and 1-methyl-4-phenylpyridium as models, Trx prevents the oxidative stress-induced apoptosis of SH-SY5Y cells. Consistent with these results, Trx conferred protection on SH-SY5Y cells against 15d-PGJ2-induced cell damage (Fig. 2).
It was proposed that the protection of oxidative cell damage by Trx might
be achieved through a direct electrophile scavenger function. To prove this
hypothesis, using a biotinylated 15d-PGJ2, we explored the
incorporation of 15d-PGJ2 into cellular proteins
(Fig. 3B) and cellular
Trx (Fig. 3C). Our
results show that biotinylated 15d-PGJ2 modifies Trx when added to
intact cells. These results raise the possibility that Trx plays an important
role in the protection against the pro-oxidant effects of the electrophilic
PG, at least at the pharmacological doses employed in most biochemical
studies. Several previous observations have indicated that PGD2 and
its J-ring metabolites might exert effects through interactions with
intracellular proteins. Narumiya et al.
(56) have shown that
radiolabeled 12-PGJ2 is actively incorporated
into cells and transferred to the nucleus, where it is associated with
proteins. Some PGs, including PGD2, PGJ2, and
12-PGJ2, have been shown to bind with high
affinity to the liver fatty acid-binding protein and intracellular protein
involved in the uptake, intracellular transport, and metabolism of free fatty
acids and their acyl-CoA esters
(57). 15d-PGJ2 has
been shown to directly inhibit NF-
B activation either by blocking
I
B kinase activity through covalent modifications of critical cysteine
residues in I
B kinase
or by interacting with cysteine residues
in the DNA-binding domain of the NF-
B subunit p65
(10,
58). Moreover, the NF-
B
p50 subunit was also identified to be a target for covalent modification by
15d-PGJ2 leading to the inhibition of DNA binding
(16). Moos et al.
(36) have also reported the
covalent modification and inhibition of Trx reductase by cyclopentenone PGs.
More recently, Olivia et al.
(59) have shown that
15d-PGJ2 induces H-Ras activation mediated by direct interaction of
15d-PGJ2 to the cysteine residue (Cys-184) of H-Ras. This and our
observations suggest cyclopentenone PGs could induce redox alteration due, at
least in part, to the inhibition of the Trx-dependent regulatory systems.
The covalent binding of 15d-PGJ2 to Trx was examined upon in vitro incubation of hrTrx with biotinylated 15d-PGJ2. The observations (Fig. 4) that the biotinylated 15d-PGJ2 was significantly incorporated into hrTrx and that the exposure of hrTrx to the biotinylated 15d-PGJ2 resulted in the loss of cysteine residues confirms that 15d-PGJ2 is a chemically reactive species capable of alkylating nucleophilic sites in the protein Trx under physiological conditions. In this study, we have used a combination of MALDI-TOF MS and ESI-LC/MS (ESI-LC/MS/MS) to elucidate the mechanism for modification of Trx by 15d-PGJ2. The MADL-TOF MS analysis of 15d-PGJ2-treated hrTrx showed that the reaction of Trx with 15d-PGJ2 resulted in the formation of at least two adducts, the major of which is a monoadduct (Fig. 5). The 15d-PGJ2-Trx adduct was further digested with trypsin, and the peptides (Tp-1 and Tp-2), containing the 15d-PGJ2-cysteine adduct, were sequenced by ESI-LC/MS/MS. Among two catalytic site cysteine residues (Cys-32 and Cys-35) and three other cysteine residues (Cys-62, Cys-69, and Cys-73) in human Trx, the sequencing analysis showed the site of modification to be exclusively at Cys-35 and Cys-69 (Figs. 6, 7, 8). These data suggest that 15d-PGJ2 may not be randomly incorporated into free SH groups but instead may alkylate cysteine groups in specific environments. The preference for alkylation of Cys-35 to Cys-32 was unexpected, because (i) the active site of Trx is known to have the cysteines on a protruding loop with Cys-32 more exposed than Cys-35 (60), and (ii) Cys-32 has a lower pKa than Cys-35 (61). Indeed, Erve et al. (62) showed that S-(2-chloroethyl)glutathione exclusively alkylates Cys-32 with no alkylation at Cys-35. Although the detailed mechanism for this preferential alkylation of Cys-35 by 15d-PGJ2 remains unclear, the modification of one of the two active site cysteines may be directly associated with the abolishment of its redox regulatory functions, because Cys-35 is critical to releasing the reduced protein substrate followed by the formation of a disulfide linkage with Cys-32. On the other hand, the non-catalytic cysteine residues have also been reported to undergo covalent modification. Haendeler et al. (63) showed recently that Trx is S-nitrosylated on Cys-69, which is located between an acidic and a basic amino acid in the proposed consensus motif for S-nitrosylation (64, 65). It has been suggested that S-nitrosylation on Cys-69 is involved in an anti-apoptotic mechanism of Trx that differs from the anti-apoptotic mechanisms mediated by the binding of proapoptotive proteins to Cys-32 and Cys-35 (63). Because this S-nitrosylation is required for scavenging ROS and for preserving the redox regulatory activity of Trx, the 15d-PGJ2 modification of Cys-69 may result in an increase in the formation of ROS. This may be associated with our previous finding (14) that 15d-PGJ2 and other cyclopentenone-type PGs can potently induce intracellular oxidative stress in SH-SY5Y human neuroblastoma cells.
In summary, our results identified the Trx as a molecular target for the covalent modification by 15d-PGJ2, providing a biochemical basis for the redox alteration by cyclopentenone PG. In addition, we identified the two target cysteine residues, Cys-35 and Cys-69, of the 15d-PGJ2 modification within Trx. The Trx modification by 15d-PGJ2 may be one of the mechanisms by which 15d-PGJ2 induces intracellular oxidative stress and neuronal cell death.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Fax: 81-52-789-5741. E-mail: uchidak{at}agr.nagoya-u.ac.jp
1 The abbreviations used are: PG(s), prostaglandin(s); 15d-PGJ2,
15-deoxy-12,14-PGJ2; Trx, thioredoxin; ELISA,
enzyme-linked immunosorbent assay; ROS, reactive oxygen species; DCFH-DA,
2',7'-dichlorodihydrofluorescein diacetate; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HRP, horseradish
peroxidase; PBS, phosphate-buffered saline; DTNB,
5,5'-dithiobis(2-nitrobenzoic acid); hrTrx, human recombinant Trx; ECL,
enhanced chemiluminescence; HPLC, high performance liquid chromatography; TBS,
Tris-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization
time-of-flight; MS, mass spectrometry; ESI-LC, electrospray ionization-liquid
chromatography.
2 K. Itoh, M. Mochizuki, Y. Ishii, T. Shibata, Y. Kawamoto, T. Ishii, V.
Kelly, K. Sekizawa, K. Uchida, and M. Yamamoto, submitted for publication.
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ACKNOWLEDGMENTS |
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