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J. Biol. Chem., Vol. 275, Issue 34, 26556-26565, August 25, 2000
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From the Department of Pharmacology, Case Western Reserve
University, School of Medicine, Cleveland, Ohio 44106-4965
Received for publication, May 15, 2000, and in revised form, June 8, 2000
Oxidative stress broadly impacts cells,
initiating regulatory pathways as well as apoptosis and necrosis. A key
molecular event is protein S-glutathionylation, and
thioltransferase (glutaredoxin) is a specific and efficient catalyst of
protein-SSG reduction. In this study 30-min exposure of H9 and Jurkat
cells to cadmium inhibited intracellular protein-SSG reduction, and
this correlated with inhibition of the thioltransferase system,
consistent with thioltransferase being the primary intracellular
catalyst of deglutathionylation. The thioredoxin system contributed
very little to total deglutathionylase activity. Thioltransferase and
GSSG reductase in situ displayed similar dose-response
curves (50% inhibition near 10 µM cadmium in
extracellular buffer). Acute cadmium exposure also initiated apoptosis,
with H9 cells being more sensitive than Jurkat. Moreover, transfection
with antisense thioltransferase cDNA was incompatible with cell
survival. Collectively, these data suggest that thioltransferase has a
vital role in sulfhydryl homeostasis and cell survival. In separate
experiments, cadmium inhibited the isolated component enzymes of the
thioltransferase and thioredoxin systems, consistent with the vicinal
dithiol nature of their active sites: thioltransferase (IC50 Thioltransferase (glutaredoxin) is a member of the
TDOR1 enzyme family, which
also includes thioredoxin and their corresponding reductase enzymes
GSSG reductase and thioredoxin reductase, respectively. Thioltransferase is a 12-kDa cytosolic protein that has been
characterized in vitro as a specific catalyst for the
reduction of protein-glutathionyl-mixed disulfides (protein-SSG)
(1-5). The reaction catalyzed by thioltransferase is also selective
for GSH as the reducing substrate (3). This thiol-disulfide interchange
reaction is likely crucial for maintaining intracellular thiol status
(2, 6). Under normal conditions the intracellular milieu is
predominately reducing (typical GSH/GSSG ratio Cysteine residues can be modified reversibly in other ways that also
can affect the activities of proteins. Examples include sulfenic acids
and intra- or intermolecular disulfides. Thioltransferase does not
catalyze reduction of these oxidized forms unless they react first with
GSH to form protein-SSG (2). However, thioredoxin has been reported to
catalyze reduction of these sulfhydryl modifications (4, 15, 20-22),
displaying preferential reduction of intramolecular disulfides and
protein-sulfenic acids compared with intermolecular disulfides and
protein-SSG (15, 20). In contrast to thioltransferase, thioredoxin is
neither an efficient nor selective catalyst for the reduction of
protein-SSG (1, 4, 15). The different substrate selectivities of
thioltransferase and thioredoxin suggest that the two TDOR enzyme
systems may contribute synergistically to sulfhydryl homeostasis.
Cadmium is a heavy metal that exhibits both acute and chronic toxicity.
In cases of severe acute exposure, death can result in a few days and
long term exposure to lesser amounts can result in damage to kidney,
lung, and bone tissue (23). Cadmium also triggers stress-like responses
in various signaling cascades (24), including modulation of the
regulation of the tumor suppressor gene p53, various
cytokines, and proteins like metallothionine and glutathione
synthetase. Cadmium has no known beneficial effects in the human body,
but it is used extensively in the production of human consumables like
batteries, plastics, various pigments, and metal coatings. Although
Cd+2 has been implicated as a pro-oxidant, it differs from
other pro-oxidant metal ions like Cu+1 and
Fe+2, because it does not participate in redox cycling
(23). This implicates direct binding of cadmium to critical cellular
components as the mechanism of toxicity, and proteins with vicinal
disulfides are expected to be particularly sensitive to inactivation by
cadmium (25).
Recently it was reported (26) that short term incubation of HT4
neuronal cells with cadmium led to an increase in the level of cellular
protein-SSG. Because cadmium is not a direct oxidant, we hypothesized
that protein-SSG accumulation was likely due to inhibition of
thioltransferase, the expected catalyst of protein-SSG deglutathionylation in cells (2, 4, 15, 19). To test this hypothesis we
examined the effects of cadmium exposure on H9 and Jurkat cells, two
T-cell-derived lines that have been extensively studied for their
responses to oxidative stress.
Here we report that cadmium treatment of cells is associated with
specific coordination to the TDOR proteins, all of which contain
vicinal cysteine residues. This coordination leads to potent inhibition
of the thioltransferase system in situ, and this effect
corresponds to a concomitant decrease in the rate of cellular
protein-SSG dethiolation. Although the enzymes of the thioredoxin
system are also sensitive to cadmium inhibition in vitro,
the thioredoxin system does not contribute significantly to cellular
protein-SSG reduction. Together these data indicate that the
thioltransferase system is the primary mechanism for homeostatic
deglutathionylation of protein-SSG in cells. In addition, acute
treatment of cells with cadmium in the concentration range that
inhibits thioltransferase leads to apoptosis, and cells transfected with the antisense cDNA of thioltransferase do not survive
selection. These data suggest that thioltransferase is necessary for
cell survival and that it plays a role in initiation of apoptosis.
Materials--
GSSG reductase (bovine and yeast) was obtained
from Sigma. Thioredoxin reductase (bovine and Escherichia
coli) was purchased from Sigma and American Diagonstica
(Greenwich, CT). E. coli thioredoxin was obtained from
Calbiochem. NADPH was obtained from Roche Molecular Biochemicals.
L-Cysteinyl-glutathione disulfide was purchased from
Toronto Research Chemicals, Inc. Lipoamide disulfide
(DL-6,8-thioctic acid amide) was obtained from Sigma. RPMI
1640 medium, fetal bovine serum, penicillin-streptomycin mix,
glutamine, trypan blue, Lipofectin, and Lipofectamine were from Life
Technologies, Inc. All other chemicals were reagent grade from standard
sources. Dr. David Davis (National Institutes of Health) generously
donated the Jurkat cells (clone E6-1), and the H9 cells and K562 cells
were obtained from the American Type Culture Collection. pRep4 and
pRep10 expression vectors were from Invitrogen. Sephadex G-75 gel
filtration resin was obtained from Amersham Pharmacia Biotech. The
micro BCA protein assay and Slide-A-Lyzer dialysis cassettes
were obtained from Pierce. YM 30 microconcentrator membranes were from
Millipore. Tran35S-label ([35S]methionine
plus [35S]cysteine) were obtained from ICN
Biochemicals, and [35S]GSH was obtained from NEN Life
Science Products.
Enzymes--
Recombinant human thioltransferases (wild type and
mutant) were expressed and purified from E. coli as
described previously (5, 27). The thioltransferase enzymes and bovine
GSSG reductase were diluted in PGN buffer (100 mM
sodium/potassium phosphate, pH 7.5, 0.5 mM GSH, and 0.2 mM NADPH) for inhibition experiments. Purified bovine
thioredoxin was obtained via stepwise chromatography as described
previously (28); frozen aliquots of the partially purified enzyme were
thawed and eluted from the final DEAE column with PGN buffer containing
0.5 M NaCl. To remove EDTA contained in the manufacturer's
buffer system, aliquots of the stock solution of bovine thioredoxin
reductase were diluted in PGN buffer to 0.09 mg/ml and reconcentrated
with a microconcentrator with a YM 30 membrane (Millipore). This
procedure was repeated twice. The final concentrate was diluted to 0.9 mg/ml in PGN buffer.
Preparation of
BSA-SSG[35S]--
BSA-SSG[35S] was
prepared as described previously (3), with the following modifications.
After the reaction of S-carboxymethyl-BSA with
N-succinimidyl pyridyl bis(3,3'-dithiopropionate) was
quenched with glycine, BSA was separated from small molecules by
dialysis against 100 mM sodium phosphate, pH 7.0, overnight
with two changes of buffer. The modified BSA was then treated with 4 mM [35S]GSH for 1 h at room temperature.
The resulting BSA-SSG[35S] product was separated from
[35S]GSH as described previously, and it typically had
Cell Maintenance--
H9, Jurkat, and K562 cells were grown in
RPMI 1640 medium supplemented with 15% fetal bovine serum (10% for
K562 cells), penicillin and streptomycin (50 units/ml and 50 µg/ml,
respectively), and 2 mM glutamine. The cells were
maintained between 1 × 105 and 1 × 106 cells/ml in a humidified 37 °C incubator containing
5% CO2 for no more than 20 days. The cells were passed
every 2-3 days.
Cell Exposure to Cadmium--
Cells in log phase growth (6 × 105 to 1 × 106, within 48 h of
passage) were centrifuged and resuspended in phosphate-buffered saline
(PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM sodium phosphate, 1.4 mM potassium
phosphate, pH 7.4) to a density of 7 × 105 cells/ml.
These cells were treated by adding 100-µl aliquots of concentrated
cadmium solutions in deionized water to 5 ml of the cells in PBS, so
that final cadmium concentrations were between 0 and 100 µM. Controls (100 µl of H2O only) were
performed in parallel. The cells were incubated at 37 °C at 5%
CO2 for 30 min. Cadmium chloride and cadmium acetate gave
equivalent results.
Cell Lysates--
Cells (which settled during treatment) were
resuspended in a minimal amount (usually 1 ml) of one of the various
treatment solutions and centrifuged, and the resulting supernatant was
removed by suction. Cell pellets were resuspended in lysis buffer, pH 7.4 (adapted from Ref. 29) (50 mM Tris-HCl, 2.2 mM sodium phosphate, 0.7 mM potassium
phosphate, 0.75% Triton X-100 (v/v), 369 mM NaCl, 1.35 mM KCl, 2 µl/ml dimethyl sulfoxide, 10 µg/ml leupeptin,
20 µg/ml aprotinin, and 25 µM p-nitrophenyl,
p'-guanidinobenzoate), in the absence or presence of 50 mM NEM. NEM was added to alkylate remaining free
sulfhydryls when the extracts were being analyzed for protein-SSG.
Protein content was assessed with the BCA assay using BSA as standard.
Assay of the Thiol-disulfide Oxidoreductase Activities in Cell
Lysates--
Lysates of control cells and cadmium-treated cells were
analyzed for thioltransferase activity with the standard radiolabel assay (3), which monitors time-dependent release of
radioactivity from BSA-SSG[35S]. Aliquots of lysate and
PGN buffer with or without yeast GSSG reductase (2 units/ml, final)
were warmed separately to 30 °C, mixed, and then an aliquot of
BSA-SSG[35S] (0.1 mM, final) was added to
initiate the reaction (total volume 0.5 ml). Aliquots of the resultant
reaction mixtures (30 °C) were precipitated with ice-cold
trichloroacetic acid (10%, final) at 0.5, 1, 2, and 3 min. After
centrifugation, the supernatants were analyzed for 35S by
scintillation counting with
Cellular thioredoxin activity was assayed using the radiolabel assay
described for thioltransferase with the following modifications: GSH
was omitted, GSSG reductase was either omitted or replaced with
thioredoxin reductase (3.6 µg/ml, final), and time points were taken
at 0.25, 15, 30, and 60 min. Addition of 5 mM EDTA to the
reactions did not alter the results.
Standard spectrophotometric assays were used to determine the cellular
activities of GSSG reductase (30) and thioredoxin reductase (31).
Lysates (30 µl) and PGN buffer (for GSSG reductase) or PGN buffer
without GSH (for thioredoxin reductase) were warmed separately to
30 °C (5 min). The two components were mixed, and the reactions were
initiated with substrate (1 mM final for GSSG or
5,5'-dithiobis(nitrobenzoic acid), respectively). GSSG reductase activity was monitored for 5 min at 340 nm, and thioredoxin reductase activity was followed for 8 min at 405 nm in separate assays. The rates
are presented as nanomoles of product/min/mg of cell protein. Although
typical procedures include EDTA in the assay mixtures, we purposely
omitted EDTA to avoid competitive chelation of cadmium. Instead buffers
were routinely pretreated with Chelex (0.1 g/ml) that had been washed
thoroughly with deionized water (18-megohm resistance).
In Vitro Inhibition of the Thiol-disulfide Oxidoreductase Enzymes
by Cadmium--
Cadmium-mediated inhibition of the TDOR enzymes was
tested in PGN buffer treated with Chelex (as above). GSH was included in all of the inhibition reactions, because it is naturally present in
the intracellular environment. Because 5,5'-dithiobis(nitrobenzoic acid) reacts with GSH immediately, thioredoxin reductase
activity was assayed with lipoamide, a known substrate for thioredoxin reductase (31) that is not rapidly reduced by GSH.
All four TDOR enzymes were tested similarly for inhibition by cadmium.
Concentrated PGN buffer, containing the respective enzymes, and stock
solutions of cadmium in water (or water alone) were prewarmed (5 min)
separately to 30 °C. Then 30 µl of the cadmium solution (or water)
was added to the enzyme solutions to achieve final cadmium
concentrations of 0-200 µM. The reactions were
immediately initiated with the appropriate substrate (final volume 200 µl). Each enzyme was tested with seven concentrations of cadmium
(0.1-200 µM) encompassing the full range of inhibition for each enzyme.
Activity of thioltransferase (wild type or mutant) in the absence or
presence of cadmium was assayed using both spectrophotometric and
radiolabel assays (3). The spectrophotometric assay was adapted for use
with a 96-well plate reader (Thermomax, Molecular Devices), and yeast
GSSG reductase was used as coupling enzyme. Because the yeast GSSG
reductase was inhibited at higher cadmium concentrations, separate
controls (minus thioltransferase) were performed for each concentration
of cadmium. In the radiolabel assay GSSG reductase was omitted to
obviate this complication, and time points were taken at 0.25, 0.5, 1.0, and 1.5 min. The two assays yielded identical results.
Mammalian (bovine) GSSG reductase was analyzed for activity in the
absence or presence of cadmium as described above for analysis of GSSG
reductase in cell lysates.
Activity of mammalian (bovine) thioredoxin reductase in the absence or
presence of cadmium was assayed using 1 mM lipoamide (stock
solution 10 mM in 20% ethanol) as the substrate.
Activity of mammalian (bovine) thioredoxin in the absence or presence
of cadmium was analyzed as previously (32) using insulin as the
substrate, except that DTT was diminished from 2 to 0.5 mM
to decrease potential cadmium chelation by DTT. To estimate the
magnitude of this DTT effect, inhibition of thioredoxin reductase by
cadmium was tested (as above) in the absence and presence of 0.5 mM DTT.
Cadmium-mediated Inactivation of Oxidized
Thioltransferase--
Thioltransferase (wild type) was pretreated with
or without cysteine-SSG (50 mM) in 100 mM
potassium phosphate buffer (pH 7.5) for 10 min at 30 °C.
Thioltransferase samples so treated were tested for sensitivity to IAA
(300 µM) and cadmium (20 µM) in comparison
to control samples run in parallel. The reaction mixtures were
incubated for 10 min at 30 °C, then tested for activity (see above)
by dilution into PGN buffer containing yeast GSSG reductase so that the
final concentrations of IAA and cadmium in the assay mixture did not
exceed 9 and 0.3 µM, respectively. These concentrations
of IAA and cadmium were confirmed separately to have no substantial
effect on the coupling enzyme yeast GSSG reductase (Ref. 33 and the
present study).
Radiolabeling of Cellular Glutathione--
The glutathione pool
in H9 or Jurkat cells was radiolabeled by adapting a previously
described procedure (4, 34). Cells (3 × 107) were
incubated in serum free medium (10 ml) with cycloheximide (50 µg/ml)
at 37 °C for 30 min to inhibit protein synthesis, allowing the
radiolabel to be incorporated predominately into cellular GSH. The
extent of inhibition of protein synthesis was calculated by comparing
the amount of radiolabel incorporated in cells treated with and without
cycloheximide. The incorporation of 35S radiolabel into
proteins from cycloheximide-treated cells was <2% of the control
treated cells, i.e. >98% inhibition.
After treatment with cycloheximide, all but 1.4 ml of the serum free
medium was removed and 1.4 ml of PBS containing cycloheximide (50 µg/ml) was added, diluting the cystine and methionine
contained in RPMI medium 2-fold to 100 and 50 µM,
respectively. Approximately 200 µCi (
The use of 100 µCi of [35S]cysteine (instead of
Tran35S-Label) resulted in a higher percentage of
DTT-releasable radiolabel (see definition below) associated with
cellular proteins. This improved the ratio of disulfide-associated
[35S]GSH to total protein radioactivity without altering
any of the experimental results (i.e. improved
signal-to-background ratio).
Analysis of Protein-SSG Content--
After dialysis to remove
excess [35S]GSH, equal amounts of cell protein were added
to two reaction mixtures, one with 10 mM DTT in water and
the other with water alone. These reaction mixtures were incubated at
37 °C for 30 min and then precipitated with ice-cold 10%
trichloroacetic acid. The supernatants were analyzed for released
radioactivity. The percentage of DTT-releasable radiolabel (which
corresponds to [35S]GSH) was calculated by subtracting
the radioactivity in the supernatants of samples treated without DTT
from the radioactivity in the supernatants of corresponding samples
that were treated with DTT. This number was divided by the total amount
of protein-associated radioactivity. DTT-releasable radiolabel is
equated with [35S]GSH based on analogy to many previous
studies. Thus, under comparable conditions it was demonstrated (8, 10,
11, 19) that protein-SSG[35S] is the predominant ( Cell Growth, Death, and Apoptosis--
Cadmium treatment (in 5 ml of PBS) was carried out for 30 min as described above. However,
prior to harvesting the cells, 4 ml of PBS was removed from the cells
and 4 ml of fresh medium was added back. The cells were returned to the
incubator, and cell samples (0.5 ml) were taken at 4, 8, 12, and
24 h, treated with trypan blue, and counted with a hemocytometer.
Results are presented as total cell counts and live cells (percentage
which excluded trypan blue). To test for apoptotic cells at 12 and
24 h, 2 × 105 cells (in 25 µl of medium) were
either stained directly with acridine orange and ethidium bromide (each
100 µg/ml) or with propidium iodide as described previously (35).
Approximately 1.5 × 107 cells were centrifuged, fixed
with ethanol, treated with citric acid buffer (0.1 M, pH
7.8), and stained with propidium iodide. The cells were then analyzed
with an EPICS XL-MCL Flow Cytometer (Coulter Corp., Miami, FL) in the
Facility at Case Western Reserve University. Propidium iodide was
excited at 488 nm, with an air-cooled argon ion laser operated at 15 milliwatts, and emission was detected with a 630-nm band pass optical
filter. Data acquisition and determination of the apoptotic fraction
were performed with XL System 2 (version 3.0) software (Coulter Corp.).
DNA content signals were linearly amplified. The integrated and peak
DNA signals were used for aggregate discrimination. All samples were
filtered with a 53-µm mesh prior to analysis. Regular cell cycle
distribution was observed in control cells, but in the treated samples
a sub G0/G1 peak was observed, indicative of
apoptosis (36).
Transfection--
The hygromycin-resistant, constitutively
active expression vectors pRep4 and pRep10 were modified by insertion
of thioltransferase cDNA (27) into the multicloning region
(identical but reversed in the two plasmids) between the
BamHI and HindIII sites to construct sense (pRep
10) and antisense (pRep 4) plasmids. Transfection was accomplished with
Lipofectamine using manufacturer's protocols, and cells were
photographed at 6 weeks. K562 cells transfected with sense cDNA
were selected for 3 weeks with hygromycin, allowed to expand for 1 week, and then frozen in liquid nitrogen at 2 × 106
cells/ml. Clones were obtained from these frozen stocks by growing them
to 5 × 104 cells/ml and then serially diluting back
to 2.5 cells/ml in medium with 300 units/ml hygromycin. This cell
mixture was distributed into a 96-well plate (300 µl per well). Two
days later, wells with a single cell (observed by light microscopy)
were monitored daily and maintained in the presence of medium until
they were passed into larger flasks. A number of these clones were
analyzed for thioltransferase activity and content (Western blot analysis).
Statistical Analysis--
All experiments are presented as
mean ± S.E. Where error bars are not visible on the figures, they
are contained within the size of the data symbol. Where applicable,
Student's two-tailed t test was used to determine
statistically significant differences (WINKS 4.5 program, TexaSoft,
Cedar Hill, TX).
Cellular Activity of Thioltransferase and Thioredoxin--
Lysates
of H9 and Jurkat cells were analyzed for deglutathionylase activity of
endogenous thioltransferase and thioredoxin (Table
I). The thioltransferase activity
(GSH-dependent dethiolase activity) in both cell lines was
substantial (Table I, left column). In contrast, little if
any thioredoxin-mediated protein-SSG dethiolase activity
(i.e. GSH-independent) was detectable even with extended 60-min reaction times, addition of 5 mM EDTA, or addition
of excess thioredoxin reductase (Table I, right column).
The lack of thioredoxin-mediated deglutathionylase activity in lysates
is consistent with previous reports of much lower catalytic activity of
thioredoxin for protein-SSG substrates when compared to
thioltransferase (1, 4, 15). A full comparative kinetic analysis of the
two enzyme systems has not been reported, so we examined the relative
catalytic efficiencies
(kcat/Km) of these enzymes.
The best prototype substrate for this comparison is cysteine-SSG,
because it represents the commonality of all protein-SSG substrates
without the steric constraints imparted by location of the cysteine-SSG
moiety in the respective proteins. Accordingly, thioltransferase was
5000 times more efficient
(kcat/Km) than thioredoxin in
catalyzing deglutathionylation of cysteine-SSG (Table
II).
Inactivation of Cellular Thioltransferase by Cadmium--
To test
whether cellular thioltransferase was inactivated, H9 and Jurkat cells
were treated with cadmium (0-100 µM) for 30 min in PBS.
Cell-derived thioltransferase activity was lost in a
dose-dependent manner as reflected by the diminution in the rate of reduction of BSA-SSG[35S] as a function of
cadmium concentration (Fig.
1A). This inactivation was
irreversible, because removal of cadmium from the cell lysates by
overnight dialysis at 4 °C did not change the percentage activity remaining in the cadmium-treated samples relative to control samples dialyzed in parallel (data not shown). Cellular GSSG reductase was also
inhibited by cadmium over the same concentration range as
thioltransferase (Fig. 1B). The lack of observable cellular thioredoxin-mediated reduction of protein-SSG (Table I) indicates that
the effect of cadmium on deglutathionylation can be ascribed exclusively to the thioltransferase system.
To investigate the effect of cadmium on thioltransferase-mediated
catalysis of reduction of endogenous protein-SSG in situ, cells were pretreated with 0.5 mM
H2O2 in PBS to maximize intracellular protein-SSG formation.2 After
replacement of the H2O2-containing PBS with
complete medium, the initial rates (
The loss of in situ protein-SSG deglutathionylation activity
(Fig. 2) is consistent with cadmium-mediated inactivation of cellular
thioltransferase activity measured in vitro (Fig.
1A). The 75% inactivation (intact cells) was somewhat
different from the 90% inactivation (measured in vitro),
but this apparent discrepancy can be reconciled (see
"Discussion").
Cadmium Affects the Accumulation of Protein-SSG--
We tested
whether cadmium treatment of H9 and Jurkat cells would result in
accumulation of cellular protein-SSG as observed in mouse neuronal
cells (HT4) (26, 37). Cadmium treatment alone did not lead to a
substantial change in detectable protein-SSG in H9 or Jurkat cells
(data not shown), suggesting less basal oxidative stress in H9 and
Jurkat cells compared with HT4 cells. Nevertheless, cadmium treatment
did inactivate thioltransferase (Fig. 1A), which is
responsible for essentially all of the cellular deglutathionylase
activity (Table I). Therefore, under oxidative stress imposed by
H2O2 treatment, cadmium pretreatment would be expected to augment protein-SSG accumulation in H9 or Jurkat cells. Indeed, H2O2-induced protein-SSG accumulation
was greater in cadmium-pretreated cells (Fig.
3A).
We then tested whether cadmium pretreatment altered the pattern of
protein-SSG formation induced by H2O2. In fact,
cells treated with cadmium and H2O2 gave a very
similar array of protein-SSG[35S] bands on
SDS-polyacrylamide gel electrophoresis as did the cells treated with
H2O2 alone (Fig. 3B). This result
indicates that cadmium was not an effective competitor for the majority of glutathionylation sites on thiol-containing proteins. This observation was confirmed by densitometric measurements of the entire
lanes (see Fig. 3 legend), and it is consistent with the affinity of
cadmium for vicinal thiols versus isolated thiols that serve
as glutathionylation sites (see below). The apparent molecular masses
of the predominant glutathionylated proteins in H9 cells after maximal
H2O2 treatment (± cadmium) were 97, 64, and 45 kDa, and a 23-kDa doublet.
Relative Sensitivity of the Thiol-disulfide Oxidoreductase Enzymes
to Cadmium Inhibition in Vitro--
Because all of the TDOR enzymes
contain vicinal thiols at their active sites, we determined their
relative sensitivities to inhibition by cadmium in vitro.
GSH was included in all of the reaction mixtures to mimic intracellular
thiol status. Among the four TDOR enzymes tested, thioredoxin reductase
was the most sensitive (Fig. 4,
IC50
To test further the supposed vicinal thiol chelation mechanism of
cadmium inactivation/inhibition, the cadmium sensitivity of unaltered
thioltransferase was compared with two altered forms; namely,
(a) oxidized thioltransferase, where the active site vicinal thiols are converted to an intramolecular disulfide (C22-SS-C25), and
(b) mutant thioltransferase (C7S, C25S, C78S, C82S) where the active site vicinal thiols are now a thiol (Cys22) and
a hydroxyl (Ser25) (5). Pretreatment of wild type
thioltransferase with disulfides converts the active site to C22-SS-C25
and protects the enzyme from alkylative inactivation by iodoacetic acid
(1, 38). Therefore, thioltransferase was preincubated in the absence or presence of cysteine-SSG, followed by treatment with no agent (control), IAA, or cadmium. The oxidized thioltransferase retained full
activity, whereas the reduced (dithiol) thioltransferase was
inactivated by IAA and cadmium consistent with the mechanism of vicinal
thiol chelation for cadmium-mediated inactivation/inhibition (Table
III). The mutant thioltransferase was
10-fold less sensitive to cadmium-mediated inhibition (IC50 Effects of Cadmium Treatment on Cell Viability--
Cellular
toxicity, apoptosis, and necrosis have previously been reported as
consequences of cadmium treatment, but these responses have usually
been determined after exposures much longer than those used in the
present study. Therefore, we measured the effect of acute cadmium
exposure on cell viability. A 30-min cadmium treatment followed by
replacement of the medium resulted in inhibition of cell growth
followed by cell death as measured by trypan blue exclusion (Fig.
5). Death of cadmium-treated cells
compared with control cells was evident at 12 h for both cell
lines. However, H9 cells (Fig. 5, left panels) were more
sensitive to cadmium than were Jurkat cells (Fig. 5, right
panels).
Whether apoptosis accounted for cadmium-mediated cell death was
determined using three different methods. Nuclear staining with
acridine orange and ethidium bromide showed characteristic apoptotic
morphology in both cell types, but H9 cells were more sensitive than
Jurkat cells. H9 cells also showed clear DNA laddering, whereas Jurkat
cells showed none (data not shown). The apoptotic response to cadmium
was quantified by flow cytometry of propidium iodide-stained cells
(Table IV). By all measures H9 cells were more sensitive to cadmium-mediated apoptosis and cell death than were
the Jurkat cells.
Transfection of Cells with Antisense Thioltransferase cDNA
Impacts Cell Survival--
To manipulate the level of thioltransferase
directly, cells were transfected with sense or antisense
thioltransferase cDNA (Fig. 6). It
was expected that derivative cell lines with various amounts of
thioltransferase would be established by manipulating K562 cells
(relatively low endogenous thioltransferase) and Jurkat cells
(relatively high endogenous thioltransferase). Remarkably, in three
separate experiments with both K562 and Jurkat cells, transfection with
antisense thioltransferase cDNA was not compatible with cell
survival. Cells transfected with antisense cDNA for thioltransferase did not survive selection (Fig. 6B),
whereas cells transfected with vector alone (Fig. 6A) or
cells transfected with sense cDNA for thioltransferase all survived
selection (Fig. 6C and data not shown). K562 cells
transfected with sense cDNA for thioltransferase displayed expected
increases in deglutathionylase activity (Fig. 6C), whereas
Jurkat cells transfected with sense cDNA did not display the
expected increase (data not shown). The collective data of Figs. 5 and
6 suggest that thioltransferase is required for cell viability (see
"Discussion").
Thioltransferase and thioredoxin are relatively abundant cytosolic
enzymes distributed ubiquitously in nature and considered to play
important roles in sulfhydryl homeostasis. The two enzymes are
distinguished by different cofactors and by different substrate specificities. Thioltransferase specifically catalyzes the reduction of
glutathione-containing mixed disulfides, in particular protein-SSG. Although thioredoxin can catalyze reduction of protein-SSG substrates, it is far less efficient than thioltransferase (Table II) (1, 4, 15,
21) and would not be expected to contribute to physiological deglutathionylation. Reversal of oxidative stress-induced protein-SSG formation has been observed in numerous studies (8-11, 19, 39, 40),
but the enzymatic basis for the deglutathionylation was not addressed.
In this report the thioltransferase-mediated
(GSH-dependent) rate of BSA-SSG[35S]
reduction by cell lysates is at least 18 times greater than the
thioredoxin-mediated (GSH-independent) rate of
BSA-SSG[35S] reduction. Moreover, the time frame of
thioltransferase-mediated reduction of BSA-SSG[35S] by
cell lysates (3 min) correlates with the time frame of intracellular deglutathionylation observed for control cells shown in Fig. 2. Thus,
cellular protein-SSG dethiolase activity is essentially entirely
attributable to cellular thioltransferase activity (Table I).
Previously we found that thioltransferase accounted for all of the
hemoglobin-SSG deglutathionylase activity of human hemolysates (2, 41),
and Jung and Thomas (4) ascribed the major deglutathionylase activity
of rat hepatocytes to thioltransferase. Thus, it seems appropriate to
generalize that thioltransferase is the primary intracellular catalyst
of protein-SSG deglutathionylation.
The Effect of Cadmium on the Thiol-disulfide Oxidoreductase
Enzymes--
In the current study, cadmium treatment of cells in a
range shown previously to alter cellular functions (24) inactivated intracellular thioltransferase and its coupling enzyme GSSG reductase (Fig. 1), consistent with in vitro effects of cadmium on
these two enzymes (Fig. 4). Our results for mammalian GSSG reductase agree with those of Muller (42) but differ from another study that
reported no inhibition (43). However, the latter study included EDTA in
the assay of GSSG reductase, likely abolishing the cadmium effect.
Mammalian thioredoxin reductase is quite sensitive to cadmium according
to our in vitro data (IC50 The Mechanism of Cadmium-Enzyme Interaction--
As suggested
previously (25), the likely mechanism of cadmium-mediated
inactivation/inhibition of the TDOR enzymes is coordination with the
vicinal thiol groups contained in all four of these enzymes. Our
current data support this interpretation in several ways: 1) oxidized
thioltransferase (C22-SS-C25) was insensitive to cadmium-mediated inactivation (Table III); 2) mutant thioltransferase (vicinal cysteine replaced by serine, C25S) was fully protected against irreversible inactivation by cadmium (Table III), and it was 10-fold less sensitive to inhibition by cadmium compared with wild type enzyme; and 3) cadmium
did not alter the profile of proteins that are glutathionylated in
response to H2O2 treatment (Fig.
3B), consistent with a monothiol nature of these sites.
Because cadmium pretreatment did not alter the profile of protein-SSGs
induced by H2O2, this means that the endogenous
substrates for thioltransferase were the same in control and cadmium
pretreated cells. Hence the diminution of protein-SSG reduction by
cadmium can be attributed unambiguously to inhibition of the
thioltransferase enzyme system rather than to the alteration of substrate.
As hypothesized, the cadmium-mediated inhibition of thioltransferase
activity in situ and in vitro (Figs. 1 and 4)
correlated with cadmium-mediated inhibition of intracellular
protein-SSG dethiolation (Fig. 2). When cells in PBS were treated with
100 µM cadmium, cellular thioltransferase activity
measured in vitro was decreased by 90%. When cells in PBS
were treated with 100 µM cadmium and 0.5 mM
H2O2 and then resuspended in medium, the initial rate of intracellular deglutathionylation measured in situ was decreased by 75%. This apparent difference in inhibitory effect can reasonably be attributed to an underestimate of the initial
rate for the untreated cells and/or to differences in conditions of the
two experiments. In particular, addition of replete medium can recruit
a portion of the endogenous thioltransferase protected from cadmium
inactivation by formation of the intramolecular disulfide (C22-SS-C25)
in response to the oxidative H2O2 treatment, a
modification readily reversed by GSH. In addition, a small portion of
the net deglutathionylation in situ may be due to direct
action of GSH that is not inhibited by cadmium and cannot be assessed separately as it can be in the in vitro measurements. Thus
the in situ experiment (Fig. 2) probably reflects an
underestimation of the actual sensitivity of thioltransferase to
inactivation by cadmium.
The Involvement of Cellular Cofactors in Thiol-disulfide
Homeostasis--
Cellular thioltransferase activity can be affected
both directly (inactivation/inhibition) and indirectly (depletion of
the second substrate, GSH). Others have reported that GSH is a
necessary factor for cellular reduction of protein-SSG, and these
results are consistent with thioltransferase being responsible for
enzymatic deglutathionylation. In particular, regeneration of GSH by
NADPH and GSSG reductase (40) was reported to be the rate-limiting step
in erythrocyte reduction of protein-SSG, and this was linked to
erythrocyte glucose 6-phosphate dehydrogenase activity. Glucose 6-phosphate dehydrogenase was reported to be inhibited partially by
cadmium in hepatocytes, but it was less sensitive than GSSG reductase
(42). In another context we found that, in genetically altered CHO
cells where glucose-6-phosphate dehydrogenase was knocked out and NADPH
generation was compromised, endogenous reduction of disulfides was also
impaired (45). These altered CHO cells appeared to be devoid of
reduction activity attributable to the thioredoxin system; however,
activity attributable to the thioltransferase system
(GSH-dependent) was diminished but not abolished (45). This
distinction is likely due to the direct dependence of the thioredoxin
system on NADPH for turnover, whereas the thioltransferase system can
operate without NADPH so long as the supply of GSH is sufficient (both
for reduction of thioltransferase and formation of
glutathionyl-disulfide substrates). Two mechanisms, de novo synthesis and NADPH-dependent turnover of GSSG, maintain
cellular GSH. In the current study H2O2-treated
cells replenished with medium (Fig. 2) reduced accumulated protein-SSG
rapidly ( Cadmium and Cell Survival--
Cadmium has been reported to cause
apoptosis and necrosis depending upon the concentration and period of
exposure (usually 8 h or more) (46-48). Cadmium treatment (4-50
µM) of CEM-C12 cells, a T-cell line, in medium with 2%
serum for 18 h was associated with apoptosis (46). In another
report (47) T-cells, monocytes, and B-cells were all shown to undergo
cell death in response to cadmium treatment (0.1 µM to 10 mM) but only after 7 h or more of incubation with
cadmium. The one exception was T-lymphocytes treated for only 1 h
but with 10 mM cadmium, a very high concentration (47).
Only 20% of the T-lymphocytes were viable 24 h later; however,
apoptosis was not addressed as a mechanism of cell death. In another
study CCRF-CEM, Raji, and Molt-3 cells were treated with cadmium in
media containing 5% serum for 18 h, and all three cell lines
underwent apoptosis at low concentrations of cadmium but underwent
necrosis at higher concentrations (above 50 µM) (48).
In contrast, we examined whether a more limited cadmium exposure (30 min), which inactivates cellular thioltransferase activity (Fig.
1A), would lead to cell death or apoptosis. In H9 and Jurkat cells, 30-min cadmium treatment followed by incubation in fresh medium
for 12 or more additional hours resulted in decreased cell viability
according to trypan blue exclusion (Fig. 5). Cadmium treatment also
caused growth inhibition and inflicted irreversible damage that led to
death by apoptosis in a concentration-dependent manner
(Figs. 5 and 6 and Table IV). Thus, loss of thioltransferase correlates
with initiation of cellular apoptosis. However, Jurkat cells were less
sensitive than H9 cells to cadmium-mediated apoptosis and cell death.
This difference cannot be ascribed solely to loss of thioltransferase
activity, because Jurkat and H9 cells displayed similar
cadmium-concentration dependent losses (Fig. 1A). The basis
for different extents of apoptotic cell death in these two cell types
is unknown, possibly reflecting differences in regulation of the
cellular apoptotic machinery among different cell types. A report (49)
published after completion of the current study also showed relatively
short term (2 h) exposure to cadmium (200 µM) led to
apoptosis in U-937 cells. The authors suggested p38 mitogen-activated
protein kinase activation as a specific event in initiation of
apoptosis. Because p38 mitogen-activated protein kinase is activated by
oxidative stress (50), and cadmium inhibition of thioltransferase
exacerbates oxidative stress via interference with sulfhydryl
homeostasis, the two studies are complementary and identify mechanisms
of cadmium action that may be related sequentially and/or synergistically.
The concept of initiation of apoptosis by modulation of thiol status
has been addressed previously (51-55). Increasing the level of GSH is
associated with inhibition of apoptosis mediated by a variety of known
stimulators (56-59). Loss of GSH has also been shown to sensitize
cells to apoptotic induction, but loss of GSH was not sufficient in
itself to initiate apoptosis (52). These findings suggest that GSH acts
to protect against the modification of sensitive protein-SH groups and
further that a sulfhydryl modification is necessary to initiate
apoptosis by this pathway. Caspases, cysteine proteinases implicated as
mediators of apoptosis, were shown to be regulated by changes in redox
status (i.e. changes in GSH or thioredoxin) (54, 60), and
elevated levels of cellular thioredoxin have been correlated with
inhibition of apoptosis (61). We hypothesize that cellular apoptosis
could be initiated by adduction of sensitive sulfhydryl groups on one
or more specific proteins and that when the repair mechanisms for these
proteins (thioltransferase and thioredoxin systems) are overwhelmed an apoptotic response occurs.
Thioltransferase and Cell Survival--
To enable studies of the
cellular function(s) of thioltransferase more directly we set out to
alter the levels of thioltransferase by transfecting cells with sense
and antisense thioltransferase cDNA. K562 cells transfected in the
sense direction survived selection and showed increased levels of
thioltransferase activity (Fig. 6C) and corresponding
increases in thioltransferase protein content (data not shown). Clones
derived from such transfected K562 cells maintained high levels of
thioltransferase. However, after we froze the cells for storage and
re-thawed them for analysis, they no longer retained high
thioltransferase activity, and this led us to explore other cell types.
Because the H9 and Jurkat lines had higher levels of endogenous
thioltransferase, we attempted to diminish its content by transfection
with antisense cDNA. Jurkat cells (and K562 cells) transfected with
antisense cDNA for thioltransferase did not survive selection,
whereas the cells transfected with empty vector did survive (Fig. 6,
A and B). This suggests that loss of cellular
thioltransferase was incompatible with cell survival.
In a study by another research group (62) the levels of
thioltransferase were increased by transfection in some MCF-7 cells, but further increases were not observed in a cell line that already had
elevated amounts of endogenous thioltransferase through chemical induction. They offered the interpretation that high levels of thioltransferase might be cytotoxic. In our K562 cells increased thioltransferase activity was not harmful (Fig. 6C), and
Jurkat cells transfected with thioltransferase cDNA in the sense
direction survived selection but did not show increased amounts of
thioltransferase (data not shown). Thus, our results parallel the
findings of Meyer and Wells (62). Although high levels of
thioltransferase might be toxic in some situations, an alternative
explanation is that cells may have mechanisms of feedback regulation at
either the protein or mRNA level that limit the steady-state amount
of thioltransferase. MCF-7 cells with higher levels of thioltransferase
(62, 63) displayed resistance to adriamycin, a mediator of oxidative
stress. We interpret the correlation between increased thioltransferase and resistance to adriamycin-induced oxidative stress as indicative of
prevention of protein-SSG accumulation that could trigger apoptosis.
The process of reversible S-glutathionylation of key
proteins has many potential cellular functions (2, 15, 18, 40, 64). The
transmission of cellular signals is crucial to many cellular processes,
including differentiation, proliferation, and apoptosis. The
implication that various types of modification of sensitive sulfhydryl
groups are involved in these processes suggests that both
thioltransferase and thioredoxin contribute to cellular regulation.
Because they have different substrate preferences, they are predicted
to have different but synergistic roles. Thus, loss of both TDOR
systems (as in cadmium treatment) would make a cell especially prone to apoptosis.
Conclusions--
Cadmium is a toxic agent that inhibits all of the
enzymes of the thioltransferase and thioredoxin systems via interaction with vicinal thiols. This allowed us to show that thioltransferase is
the preponderant mechanism of protein-SSG reduction in H9 and Jurkat
cells. We also showed that acute treatment of cells with cadmium
initiates cell death by apoptosis. Loss of thioltransferase activity is
a common factor in cadmium- and antisense-mediated cell death,
suggesting that loss of thioltransferase is an event that may initiate
apoptosis. Together, these results reinforce the concept that
thioltransferase is a key intracellular player in the enzymatic
regulation of redox-sensitive proteins.
We thank Ed Reineks, Dr. Nancy Wang, Dr.
Mikko Unkila, Colleen Tagliarino, and R. Michael Sramkoski
for expert advice in analysis of apoptosis. We also thank Dr. Anthony
Berdis, Dr. George Dubyak, and Dr. Michael Maguire for critical
evaluation of this work.
*
This research was partially funded by Grant A1-36219 from
the Center for Aids Research, National Institutes of Health (to J. J. M.), by National Institute on Aging program project Grant AG15885 (to J. J. M.), and by a Veterans Administration Merit Review
grant (to J. J. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Presented in part at Experimental Biology '99, Washington, DC
(FASEB J. 13, A155).
§
To whom correspondence should be addressed: Dept. of Pharmacology,
School of Medicine, Case Western Reserve University, Cleveland, OH
44106-4965. Tel.: 216-368-3383; Fax: 216-368-3395; E-mail: jjm5@
po.cwru.edu.
Published, JBC Papers in Press, June 14, 2000, DOI 10.1074/jbc.M004097200
2
In replete medium much more
H2O2 (>5 mM) would be necessary to
obtain maximal protein-SSG formation (see Fig. 3).
The abbreviations used are:
TDOR, thiol-disulfide oxidoreductase;
BSA-SSG, bovine serum albumin
(S-carboxymethyl)-NH2-CO-CH2-CH2-S-S-glutathione;
DTT, dithiolthreitol;
GSH, glutathione;
GSSG, glutathione disulfide;
IAA, iodoacetic acid;
PBS, phosphate-buffered saline;
protein-SSG, protein-glutathionyl-mixed disulfide;
NEM, N-ethylmaleimide;
CHO, Chinese hamster ovary.
Acute Cadmium Exposure Inactivates Thioltransferase
(Glutaredoxin), Inhibits Intracellular Reduction of
Protein-glutathionyl-mixed Disulfides, and Initiates Apoptosis*
,
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1 µM), GSSG reductase
(IC50 1 µM), thioredoxin
(IC50 8 µM), thioredoxin reductase
(IC50 0.2 µM). Disruption of the vicinal
dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S
mutation) protected against cadmium, consistent with a dithiol
chelation mechanism of inactivation.
INTRODUCTION
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100), but
physiological and pathophysiological processes like aging,
cardiovascular and neurodegenerative diseases, AIDS, and cancer
chemotherapy can shift the redox balance toward an oxidizing milieu. A
crucial component of the cellular redox balance is modulation of the
thiol-disulfide status of critical cysteine residues on proteins, and a
prevalent modification of cysteine residues is reversible
S-glutathionylation of proteins. Accumulation of protein-SSG
has been reported in different cell types under a variety of oxidative
conditions (7-11). With many sulfhydryl proteins, glutathionylation is
inactivating. Examples include the transacting factor NF-1 (12),
protein tyrosine phosphatase 1B (13), and phosphofructokinase (14, 15).
With other proteins glutathionylation can be an activation event.
Examples include HIV-1 protease (16, 17) and microsomal glutathione
S-transferase (18). Contrasting effects of reversible
glutathionylation for different proteins studied in vitro
suggest that protein-SSG formation is a mechanism of regulation and/or
intracellular signal transduction (2, 6, 11, 13, 17, 19) and implicate
thioltransferase as a key player in these cellular processes.
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0.9 GS-equivalent/mol of BSA.
97% efficiency (cpm). The total rates of
deglutathionylation (slopes of [35S]GSH released
versus time) were corrected for nonenzymatic
deglutathionylation by subtracting the rate of [35S]GSH
released by GSH in the absence of cell lysate. The resultant enzymatic
rates were expressed as nanomoles of product/min/mg of cellular protein.
20 µl) of
Tran35 s-Label ([35s]methionine plus
[35s]cysteine) was added to the cell mixture. The cells
were incubated at 37 °C for 4 h, centrifuged, washed three
times, and then diluted to 7 × 105 cells/ml in PBS
for cadmium or control treatment (30 min), as described above. The
cells were sometimes treated with H2O2 and/or replete medium before cell lysates were prepared, as described above.
Lysates were dialyzed repeatedly against PBS in Slide-A-Lyzer cassettes with a molecular mass cutoff of 10,000 Da until the radioactivity in the dialysate was less than 200 cpm/ml (typically accomplished with three buffer changes over an 8- to 12-h period).
90%)
radiolabeled protein adduct formed under conditions of oxidative stress.
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Rates of BSA-SSG[35S] deglutathionylation by cell lysates
Relative catalytic efficiencies of thioltransferase and thioredoxin
with the prototype substrate cysteine-SSG
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Fig. 1.
A, inactivation of cellular
thioltransferase activity by cadmium treatment: H9 cells (dotted
bars) and Jurkat cells (solid bars) in PBS were treated
with various concentrations of cadmium for 30 min. Lysates were
prepared and assayed for protein content and thioltransferase activity
as described under "Experimental Procedures." Each experiment was
repeated at least seven times, and the activities are presented as
nanomoles/min/mg (mean ± S.E.). B, inactivation of
cellular GSSG reductase activity by cadmium treatment: H9 cells
(dotted bars) and Jurkat cells (solid bars) in
PBS were treated with various concentrations of cadmium, and the
resulting lysates were analyzed for protein content and GSSG reductase
activity as described under "Experimental Procedures." Each
experiment was repeated at least three times, and the results are
presented as nanomoles/min/mg (mean ± S.E.).
25% substrate depletion) of
deglutathionylation of endogenous protein-SSG in control (0-0.5 min)
and cadmium-treated cells (0-2 min) were found to be 0.47/min and
0.12/min, respectively (Fig. 2). This
difference corresponds to a 75% net loss of protein-SSG deglutathionylation capacity after exposure of cells to cadmium plus
H2O2 compared with H2O2
alone, however, the control rate may be an underestimate, because it is
defined only by the earliest point that could be measured.
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Fig. 2.
Time courses of intracellular
deglutathionylation of protein-SSG[35S] after treatment
with H2O2 ( 
) or cadmium and
H2O2 (
). The cellular pool of GSH was
radiolabeled as described under "Experimental Procedures," and the
cells were treated with cadmium in PBS; 20 min into the cadmium
treatment the control cells and cadmium-treated cells were further
treated with 0.5 mM H2O2 for 10 min
at 37 °C to produce intracellular protein-SSG[35S].
The cells were resuspended (an aliquot was taken as the
t = 0) and centrifuged, and the cell pellets were
resuspended in replete medium at 37 °C. Then 1-ml aliquots were
taken at the time points indicated and centrifuged, and the cell
pellets were lysed in the presence of 50 mM NEM. These
lysates were dialyzed to remove free radiolabel and then analyzed for
DTT-releasable radioactivity ([35S]GSH). The amount of
[35S]GSH released by DTT at t = 0 corresponded to 50 ± 2.5% and 50 ± 2.3% of total
protein-associated radioactivity for control and cadmium-treated cells,
respectively. The data are presented as fraction of total
protein-associated [35S]GSH (mean ± S.E.,
n = 3) compared with what was released at
t = 0 (maximum release). The initial rates were
calculated from the data for the 0- to 0.5-min time period for control
cells and the 0- to 2-min time period for cadmium-treated cells
(i.e. where substrate depletion was not greater than 25% in
either case).
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Fig. 3.
H2O2-induced
protein-SSG accumulation in H9 cells ± cadmium pretreatment.
A, [35S]GSH-radiolabeled cells were treated
with 100 µM cadmium in PBS (dotted bars) or
PBS alone (solid bars), as described under "Experimental
Procedures." The cells were then centrifuged and resuspended in
replete medium, and 1-ml aliquots were treated with the indicated
concentrations of H2O2 for 10 min, and then
centrifuged. The cell pellets were analyzed as described under Fig. 2.
The data are presented as percentage of total protein radiolabel
released by DTT (mean ± S.E., n = 3).
B, lysates of radiolabeled H9 cells treated with 0.5 mM H2O2 ± cadmium, were combined
and treated ± DTT at equivalent protein concentrations. Equal
amounts of protein (1.7 mg) were run on a 10-20% SDS-polyacrylamide
gel electrophoresis gel, transferred to a polyvinylidene difluoride
membrane, exposed to film, and finally treated with Ponceau S. Left panel, densitometric scan of membrane after staining
with Ponceau S. Right panel, densitometric scan of
autoradiograph. Densitometry measurements of the lanes on the
autoradiograph were: control = 180, control with DTT = 45, cadmium = 161, and cadmium with DTT = 34.
0.2 µM). Thioltransferase and GSSG
reductase both displayed IC50 values near 1 µM. The apparent IC50 value for thioredoxin was 40 µM in the presence of 0.5 mM DTT
(which is necessary for turnover of thioredoxin in the absence of
thioredoxin reductase). To test the potential effect of DTT on cadmium
inhibitory potency, we retested cadmium inhibition of thioredoxin
reductase in the presence of 0.5 mM DTT, and found the
cadmium concentration-response relationship shifted to the right by
about a factor of 5 (data not shown). Accordingly, the IC50
for inhibition of thioredoxin in the absence of DTT can be estimated at
8 µM, as depicted by the solid line in Fig.
4.
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Fig. 4.
Inhibition of the thiol-disulfide
oxidoreductase enzymes by cadmium. Purified thioltransferase
( ), thioredoxin (
), GSSG reductase (
), and thioredoxin
reductase () were assayed for activity in the presence of GSH and
cadmium at 30 °C as described under "Experimental Procedures."
The solid line represents the theoretical results for
thioredoxin in the absence of DTT (see text). Results are presented as
percentage activity remaining ± S.E. The control activities for
each enzyme were as follows: Thioredoxin reductase (0.33 units
ml
1), thioltransferase (1.3 units ml1),
GSSG reductase (2.0 units ml1), thioredoxin with 0.5 mM DTT (
A650 nm
min1 ml1/lag time = 18 A650 nm min2
ml1).
10 µM) compared with wild type (1 µM).
The residual inhibitory effect of cadmium on the mutant
thioltransferase is likely due to the conservative substitution of
cysteine with serine, which retains some ability to coordinate with
cadmium to form a chelate complex. In fact, this inhibition was fully
reversed upon dilution, in contrast to the wild type enzyme (Table
III), i.e. the mutant without vicinal thiols was insensitive
to irreversible inactivation.
Effects of IAA and cadmium pretreatment on various forms of
thioltransferase
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Fig. 5.
Inhibition of cell growth and cell death
after cadmium treatment. Cells were treated with cadmium for 30 min in PBS, then this solution was removed and replaced with replete
medium for the indicated times (see "Experimental Procedures").
Cell aliquots were incubated with trypan blue for 5 min, and then all
cells were counted and analyzed for trypan blue exclusion. The results
are expressed as cells/ml ( , H9; , Jurkat) and percentage of live
cells (). Left panels, H9 cells; right panels,
Jurkat cells.
Cadmium induced apoptosis
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Fig. 6.
Viability of cells transfected with
plasmids ± thioltransferase cDNA inserted in the antisense
orientation. Jurkat cells (A1) and K562 cells
(A2) transfected with pRep4 containing no insert were viable
6 weeks after selection with hygromycin. Jurkat cells (B1)
and K562 cells (B2) transfected with pRep4 containing the
thioltransferase insert in the antisense orientation did not survive
selection with hygromycin. C, K562 cells transfected in the
sense direction (pRep10) survived selection and displayed increased
thioltransferase activity, assayed as described under "Experimental
Procedures" using equivalent amounts of cell lysates. Details of
selection are described under "Experimental Procedures." Results
are representative of three separate experiments.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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0.2 µM). This result compares well with cadmium inhibition of
E. coli thioredoxin reductase (25), and it is consistent
with the reported protective effect of EDTA on mammalian thioredoxin
reductase (44). Our in vitro results suggest that the
thioredoxin system would be inhibited at lower concentrations of
cadmium in situ than those that inhibit the thioltransferase
system and intracellular protein-SSG dethiolation. This analysis
further supports the conclusion that only the thioltransferase system
is pertinent to intracellular protein-SSG dethiolation.
0.47/min, initial rate), whereas cells maintained in PBS
displayed very little reduction of protein-SSG (<0.005/min,
i.e. <1% of the rate in replete medium), indicative of GSH
deprivation. These results reflect the rate-determining step of the
thioltransferase catalytic cycle where regeneration of
thioltransferase-S from the thioltransferase-SSG
intermediate requires GSH (3). Thus, cellular protein-SSG status could
be altered by several different perturbations that impact on the
activity of the thioltransferase system, including changes in GSH
concentration, concentration of NADPH, and thioltransferase activity.
ACKNOWLEDGEMENTS
FOOTNOTES
This study was conducted by Carol A. Chrestensen in partial
fulfillment of the requirements for the Ph.D. degree, Case Western Reserve University, Department of Pharmacology.
ABBREVIATIONS
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