 |
INTRODUCTION |
The proteasome is an essential proteolytic complex in eukaryotic
cells where it is responsible for the degradation of many cellular
proteins. It plays an important role in cell-cycle regulation, cell
signaling, including apoptosis, and elimination of abnormal proteins
generated by mutation (1, 2) and oxidative damage (3-5).
In recent years, many publications have reported the reversible
S-glutathionylation of a discrete number of proteins (6). Protein S-glutathionylation seems to play an essential role
in redox regulation. This form of regulation has direct effects on both
enzyme activity and the ability of transcription and replication factors to bind DNA targets. The mechanism of protein glutathionylation has evolved in recent years from the strict belief that this event would take place solely when intracellular
GSSG1 levels increased upon
oxidative stress, with the formation of mixed disulfides between
protein Cys-SH residues and GSSG (7, 8), to the present and more
complete understanding of protein sulfhydryl chemistry and evidence
showing formation of protein-Cys-SOH derivatives (9) that are prone to
S-glutathionylation by reduced GSH (10).
Either individual enzyme activity or global cellular responses can be
rapidly controlled by the oxidation of protein-Cys-SH residues
(reviewed in Ref. 6) generating Cys-SOH, Cys-SO2H, and
Cys-SO3H acid forms (11), where Cys-SOH is susceptible to S-thionylation and reversibly reduced to Cys-SH (11-14).
Cys-SOH formation and S-glutathionylation during enzyme
catalysis and redox signaling are novel cofactors in the context of
redox regulation (14).
In a recent publication (15) it was demonstrated that the reduced and
oxidized forms of GSH modulate the chymotrypsin-like activity of
purified 20 S proteasome extracted from mammalian cells. In the present
report we show that the activity of the 20 S proteasome purified from
the yeast Saccharomyces cerevisiae is also sensitive to GSH,
though in a different way from that observed in the mammalian
proteasome. The 20 S proteasome extracted from yeast is inhibited by
reduced GSH and S-glutathionylated in vitro, as
well as in vivo, when cells are submitted to oxidative challenge. Considering that the proteasome plays important role in cell
signaling regulation by hydrolysis of many proteins involved in cascade
events of the cellular regulatory pathways, it is not surprising that
its activity may be regulated by its Cys-SH residues redox status.
 |
MATERIALS AND METHODS |
Chemicals and Reagents--
Diethylenetriaminepentaacetic acid
(DTPA), dimedone (5,5-dimethyl-1,3-cyclo-hexanedione),
dinitrophenylhydrazine, dithionitrobenzoic acid (DTNB),
N-ethylmaleimide (NEM), fluorogenic substrates
succinyl-Leu-Leu-Val-Tyr-MCA (s-LLVY-MCA) and
t-butoxycarbonyl-Gly-Lys-Arg-MCA,
-glutamylcysteine (GC), and streptavidin immobilized on 4% beaded
agarose were purchased from Sigma. The fluorogenic substrate
carbobenzoxy-Leu-Leu-Glu-MCA, the proteasome inhibitors lactacystin and
tri-leucine vinyl sulfone, and monobromobimane (mBrB) were purchased
from Calbiochem. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) was
purchased from Aldrich. All other reagents used were of analytical
grade, and the water was purified with the Milli-Q system.
Yeast Strains and Growth--
S. cerevisiae
BY4741 strain (MATa his3
1 leu2
0
met15
0 ura3
0) was obtained from Euroscarf,
Frankfurt, Germany, and RJD1144 (MATa his3
200
leu2-3,112, lys2-801 trp1
63
ura3-52
PRE1FH::Ylplac211
URA3) derived from strain JD47-13C was kindly donated by Dr.
Raymond Deshaies, Division of Biology, Caltech, Pasadena, CA. This
strain has the 20 S proteasome PRE1 subunit tagged with the FLAG
peptide sequence and a polyhistidine tail (16). Cells were cultured in
YPD medium (2% glucose, 2% peptone, and 1% yeast extract) or as
otherwise specified at 30 °C with reciprocal shaking. Log phase
cells were harvested at the optical density of the culture at 600 nm
(A600) of 0.6-0.8. Stationary phase cells were
harvested at A600 higher than 1.5.
Extraction and Purification of the 20 S Proteasome--
Yeast
cells were disrupted according to the protocol described in Ref. 16.
The 20 S proteasome from untagged strain was purified by sequential
chromatographs on DEAE-Sepharose, SephacrylTM S-400 column
(Amersham Biosciences), and a monoQ column in an HPLC system.
The purification was performed according to a method described
previously (17), except that gel filtration was used instead of a
sucrose gradient, and the monoQ column was used in the HPLC instead of
the fast protein liquid chromatography system. The 20 S proteasome core
from the RJD1144 strain with a FLAGHis6-tagged PRE1
subunit was purified by affinity chromatography or, when specified, by
immunoprecipitation. Affinity chromatography was performed using a
HiTrapTM chelating HP column (Amersham Biosciences)
attached to a P1 peristaltic pump (Amersham Biosciences) according to
the manufacturer's protocol. The proteasome was eluted from the column
with 300 mM imidazole. Active fractions from all steps of
all preparations were assayed by the degradation of the fluorogenic
peptide s-LLVY-MCA and confirmed by inhibition with tri-leucine vinyl
sulfone. Final enriched fractions were pooled, concentrated, and
rediluted twice in a Centricon CM-30 apparatus (Amicon). Purification
by immunoprecipitation was performed according to the protocol
described in Ref. 16 with the anti-FLAG® M2 affinity gel freezer-safe
antibody (Sigma) immobilized on agarose beads. The 20 S proteasome was
eluted from the agarose beads, when specified, by incubation with the
FLAG® peptide (Sigma) according to the manufacturer's protocol. The final preparations were analyzed by SDS-PAGE and by electrophoresis on
a 5% non-denaturing polyacrylamide gel according to the protocol described in Ref. 17. The three different 20 S proteasome preparations described here were used for the assays. The results shown below (in vivo and in vitro) refer to the preparations
obtained from strains containing FLAGHis6-tagged PRE1;
however, we did not observe any difference compared with preparations
from strains containing untagged PRE1 (data not shown).
Hydrolysis Assay of Fluorogenic Peptides--
20 S proteasome
(0.5-3 µg) or cell extracts (400-500 µg protein/ml) were
incubated at 37 °C in 10 mM Tris/HCl buffer, pH 7.8, containing 20 mM KCl and 5 mM
MgCl2, here referred to as standard buffer. Incubation was
started by the addition of 25-50 µM of the peptides
(other additions are specified in the legends to the figures). The
reaction was stopped by adding 4 volumes of 0.1 M sodium
borate, pH 9, containing 7.5% ethanol. Fluorescence emission was
recorded at 430 nm (excitation at 365 nm). MCA liberated from the
substrates was calculated from a standard curve of free MCA.
Oxidation of 20 S Proteasome with
H2O2--
When specified, purified 20 S
proteasome preparations were treated with H2O2.
Proteasome concentration in these experiments was typically 50 µg or
as otherwise specified, and the preparation was incubated for 30 min at
room temperature in the presence of 5 mM
H2O2 with or without 100 µM DTPA
in standard buffer. After incubation, excess
H2O2 was removed by three cycles of
centrifugation and redilution with standard buffer through Microcon
CM-10 filters (Amicon). Aliquots of the 20 S proteasome were taken for
further incubation or for hydrolysis assay after determination of
protein concentration.
Reduction of 20 S Proteasome with NaBH4--
After
isolation of the 20 S core with the anti-FLAG antibody, as described
above, the agarose beads were washed three times according to the
manufacturer's protocol. NaBH4 reduction was performed
with the 20 S proteasome bound to the anti-FLAG-agarose bead complex
incubated in the presence of 20 mM NaBH4
buffered in 50 mM Tris/HCl, pH 8.6, for 40 min at 37 °C.
After incubation, the suspension was centrifuged, the supernatant was
utilized for GSH determination (described below), and the beads were
washed twice with standard buffer. The proteolysis assay, when
specified, was performed with the 20 S proteasome still bound to the beads.
GSH Determination--
Intracellular GSH was extracted by lysing
the cell pellets in 1 volume of glass beads and 2 volumes of 3.5%
sulfosalicylic acid. The suspension was vortexed for 20 min at 4 °C
in a multifold vortex and centrifuged at maximum speed in a
microcentrifuge. This procedure was repeated twice, and the
supernatants were combined. Total GSH, as well as GSSG, was assayed
according to a protocol described previously (15). The determination
was performed by reaction with DTNB in the presence of glutathione
reductase and NADPH. Samples for GSSG measurement were incubated
previously for 1 h with NEM after adjusting the pH to 7. GSH
released from 20 S proteasome was detected as follows: 20 S proteasome
isolated by immunoprecipitation was reacted with NaBH4 as
described above. The supernatant recovered was filtered through a
Microcon CM-10 filter. The filtrate was acidified to remove remaining
NaBH4 (reaction with NaBH4 was performed at pH
8.6), and the pH was adjusted to 7 prior to the addition of DTNB,
glutathione reductase, and NADPH. GSH detection was increased in the
presence of glutathione reductase and NADPH comparing to detection in
the presence of DTNB only. GSH concentration was calculated from
standard curves of GSSG. Intracellular reduced GSH was determined by
the difference between total GSH and GSSG.
GSH-Biotin Preparation--
GSH-biotin derivative was achieved
as described in Ref. 18. GSH was reacted with sulfo-NHS-biotin
(EZ-LinkTM NHS-LC-Biotin; Pierce) at 1/1 molar ratio in 50 mM NaHCO3, pH 8.5, for 1 h at room
temperature. The reaction was stopped by the addition of 5×
excess NH4CO3 compared with initial biotin concentration.
Proteasome S-Modification by the GSH-Biotin
Derivative--
H2O2-treated or untreated 20 S
proteasome (450 µg) was incubated with 5 mM GSH-biotin
for 20 min at room temperature. Excess GSH-biotin was removed by
filtration through a Microcon CM-10 filter followed by redilution three
times in standard buffer. After washing, protein was diluted in
standard buffer, mixed with streptavidin-agarose beads (100 µl/mg
protein), and incubated for 60 min at 4 °C. The beads were washed by
four cycles of centrifugation/redilution in standard buffer containing
150 mM NaCl, and the remaining protein was eluted from the
beads by incubation for 30 min in standard buffer containing 0.1% SDS
and 10 mM DTT, followed by centrifugation at maximum speed
for 15 min. The supernatants were submitted to 12.5% SDS-PAGE after
determination of protein concentration.
Cell Viability--
Aliquots of the cell suspension were diluted
to an A600 of 0.2 immediately after the
treatments, followed by a further 5,000× redilution. Aliquots
of 100 µl of the final suspension were spread on YPD-agar plates
(five per sample) and incubated at 30 °C for 48 h. After
incubation, colonies were counted manually. Cell viability assays were
repeated at least five times.
Assay for the Protein Carbonyl Group--
Carbonyl group
formation in the proteins was determined by derivatization with
dinitrophenylhydrazine and quantified by spectrophotometry as described
in Ref. 19.
Protein Determination--
Protein concentration was determined
with Bradford® reagent (Bio-Rad).
 |
RESULTS |
Yeast 20 S Proteasomal Activity Is Inhibited by Sulfhydryl
Compounds--
Purified 20 S proteasome core isolated from S. cerevisiae was assayed for hydrolysis with the s-LLVY-MCA
substrate in the presence of GSH, GSSG, and Cys (Fig.
1). GSSG had a slight effect on 20 S
proteasomal activity at any concentration tested. However, GSH or Cys
strongly inhibited the proteasomal activity at 5 mM concentration. The GSH precursor GC also inhibited chymotrypsin-like activity in a pattern similar to that obtained by Cys treatment (results not shown). The trypsin-like activity determined by the hydrolysis of the fluorogenic substrate
t-butoxycarbonyl-Leu-Lys-Arg-MCA was 50% decreased in the
presence of 10 mM GSH (results not shown). The trypsin-like
specific activity (µmol MCA/min/mg) represented less than 5% of the
chymotrypsin-like activity measured under the experimental conditions
described here (the same protocols were used for both
determinations). The peptidylglutamyl-hydrolizing activity assayed by
the hydrolysis of the fluorogenic peptide carbobenzoxy-Leu-Leu-Glu-MCA
was not affected by any of the sulfhydryl compounds tested (GSH, GC, or
Cys) at any concentration or by GSSG (result not shown).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Chymotrypsin-like activity of the 20 S
proteasome extracted from yeast determined in the presence of GSH,
GSSG, and Cys. The 20 S proteasome (1-3 µg/ml) isolated from
S. cerevisiae, as described under "Materials and
Methods," was preincubated in standard buffer for 10 min at room
temperature in the presence of 0-5 mM Cys, GSH, or GSSG,
followed by a further 1-h incubation at 37 °C with 10 µM s-LLVY-MCA. Results are expressed as means ± S.D. of
four independent experiments.
|
|
One question raised by these results was how the reduced form of GSH
and other thiols tested (Cys and GC), but not GSSG, inhibited 20 S
proteasomal activity. Our working hypothesis was that Cys-SH residues
in the 20 S proteasome structure are probably oxidized to the Cys-SOH
form, susceptible to S-glutathionylation by GSH, as
described elsewhere (6, 20), but not by GSSG, according to the
reaction,
To test this hypothesis, purified 20 S proteasome preparations
were incubated in the presence of 5 mM
H2O2 and 100 µM DTPA. The iron
chelator DTPA was used to prevent the Fenton reaction and consequently
the generation of the very reactive hydroxyl radical, which may produce
nonspecific protein oxidation, in addition to iron-catalyzed thiol
oxidation and generation of several protein-sulfur derivatives (21).
Thus, in the absence of iron or another transition metal, sulfhydryl
oxidation to Cys-SOH may prevail, though further oxidation of Cys-SOH
to Cys-SO2H and Cys-SO3H is expected (11). According to our results, 20 S proteasome treatment with
H2O2 in the presence of DTPA decreased
chymotrypsin-like activity to 80% of the original level (Fig.
2A), whereas when
H2O2 treatment was performed in the absence of
DTPA the activity was reduced to 60% (data not shown). When 20 S
proteasome was pretreated with H2O2 plus DTPA
the chymotrypsin-like activity was much more affected by GSH (Fig.
2A). In fact, after H2O2/DTPA
pretreatment, inhibition by GSH occurred at concentrations as low as
0.01 mM (35% inhibition), and GSH at 1 mM
promoted stronger inhibition (60%; see Fig. 2A) than that
verified without pretreatment (Fig. 1). In contrast, GSSG did not
affect 20 S proteasome activity (Fig. 2A). These results are
in agreement with our hypothesis in the reaction shown above.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 2.
A, H2O2/DTPA
pretreatment of 20 S proteasome sensitizes chymotrypsin-like activity
to GSH inhibition. 20 S proteasome was pretreated with
H2O2 in the presence of DTPA, as described
under "Materials and Methods." Aliquots of
H2O2-treated samples (1-3 µg) were taken and
incubated with GSH or GSSG, as described in the legend for Fig. 1,
followed by a hydrolysis assay with s-LLVY-MCA. B, DTT
preincubation followed by oxidation with
H2O2/DTPA increases 20 S core sensitization to
GSH inhibition. 20 S proteasome (100 µg) was preincubated for 30 min
at room temperature in standard buffer in the presence of 10 mM DTT. After incubation, DTT was washed out by filtration
through Microcon CM-10 filters. Protein recovered from the
filter was treated with H2O2/DTPA, as described
under "Materials and Methods." Aliquots of
H2O2-treated samples (1-3 µg) were taken and
incubated with GSH or GSSG, as per the legend for Fig. 1, followed by a
hydrolysis assay with s-LLVY-MCA. Results are represented as percentage
of control samples (no treatment) set as 100 and are expressed as
means ± S.D. of four independent experiments.
|
|
In contrast to H2O2, DTT treatment enhanced
proteasomal activity by about 15-20% (Fig. 2B). Moreover,
it sensitized the proteasome to GSH incorporation after
H2O2 treatment. When 20 S proteasome samples
were treated with 10 mM DTT, followed by incubation with H2O2/DTPA, the chymotrypsin-like activity was
even more sensitive to GSH when compared with samples not preincubated
with DTT (Fig. 2A). In this condition, proteasomal activity
was decreased to 50 and 5% in the presence of 0.5 or 1 mM
GSH, respectively (Fig. 2B), whereas when the same GSH
concentrations were employed without DTT pretreatment,
chymotrypsin-like activity was decreased only to 60 and 40%,
respectively (Fig. 2A). Cys-SOH already present in 20 S
proteasome was probably reduced to Cys-SH by DTT, with the consequent
prevention of hyperoxidation of Cys-SOH to Cys-SO2H or
Cys-SO3H by H2O2 treatment.
As expected according to our hypothesis described in the reaction shown
above, when the 20 S core was pretreated only with DTT, no alteration
by GSH was observed in its activity (Fig. 2B). Also,
proteasome reduced by DTT was not inhibited by GSSG (result not shown)
as could be expected, because GSSG is able to react with sulfhydryl
groups to form mixed disulfides (7, 8). Reduced Cys-20 S might be
prevented from reacting with GSSG by structural constraints imposed by
nearby groups.
Proteasomal Activity Is Inhibited by Cys-SH and Cys-SOH
Reactants--
To demonstrate that modification of Cys-20 S core
residues is responsible for the inhibition of chymotrypsin-like
activity, we next tested this activity in the presence of Cys-SH
and Cys-SOH reactants, such as NBD, dimedone, and NEM
(Table I). We observed that
chymotrypsin-like activity was inhibited 40 and 30% by 50 µM NBD and 1 mM NEM, respectively. NEM
alkylates Cys-SH whereas NBD is incorporated into both Cys-SH and
Cys-SOH. This might be, at least in part, the reason why NBD is more
potent than NEM in terms of chymotrypsin-like activity inhibition.
After inhibition by NBD, 20 S proteasomal activity was recovered by
incubation with 5 mM DTT (result not shown). Because DTT
treatment leads to NBD release (9), this result indicates that
inhibition was promoted by Cys conjugation.
View this table:
[in this window]
[in a new window]
|
Table I
Effect of Cys-SH and Cys-SOH reactants on proteasomal activity
The 20 S proteasome (1-3 µg/ml) was preincubated, as per Fig. 1, for
30 min at room temperature in the presence of NBD, NEM, or dimedone, at
the indicated concentrations, followed by a further 1-h incubation at
37 °C with 10 µM s-LLVY-MCA.
H2O2/DTPA-treated samples, prior to the hydrolysis
assay or dimedone incubation, samples were treated with 5 mM H2O2 and 100 µM DTPA, as
described under "Materials and Methods," followed by incubation
with dimedone and the assay with the fluorogenic substrate. The
s-LLVY-MCA hydrolysis assay is described under "Materials and
Methods." Results are means ± S.D. of three independent
experiments.
|
|
The specific Cys-SOH reagent dimedone (9) produced low inhibition when
compared with the former reagents. Dimedone promoted 23% inhibition
only at 10 mM concentration. On the other hand, when
purified 20 S proteasome preparations were pretreated with H2O2/DTPA prior to incubation with dimedone, we
observed increased proteolytic inhibition compared with the inhibition
observed in the absence of H2O2/DTPA
pretreatment followed by dimedone incubation (results shown in
italics in Table I). Upon H2O2
pretreatment, 10 mM dimedone decreased chymotrypsin-like
activity to 50% of that observed in H2O2
control samples.
Taken together, the results reported thus far indicate that any group
located in Cys residues of the 20 S core decreases its hydrolytic
activity, at least the chymotrypsin-like activity, which is considered
the strongest of its activities (1). It seems that Cys residues in the
20 S proteasome must be reduced as much as possible to allow maximum
activity. Nevertheless, these residues appear to oxidize easily to
Cys-SOH.
Cys-SOH Formation and S-Glutathionylation--
To demonstrate that
Cys residues in the 20 S proteasome structure are oxidized to Cys-SOH,
20 S proteasome preparations were incubated with NBD. This compound
reacts with Cys-SH, as well as with Cys-SOH. NBD adducts of Cys-SOH and
Cys-SH can be distinguished by their spectra (9). The
proteasome-Cys-S(O)-NBD adduct was generated by
20 S proteasome pretreatment with H2O2/DTPA
followed by NBD incubation (Fig. 3,
dashed line spectrum). This adduct showed maximum absorbance
at 345 nm whereas the purified 20 S core not oxidized by
H2O2 yielded the NBD adduct with a maximum absorbance at 420 nm (Fig. 3, solid line spectrum).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
NBD-modified 20 S proteasome UV-visible
spectra. The Cys-S(O)-NBD conjugate (dashed
line) was generated by incubating denatured 20 S proteasome (40 µg) in standard buffer with H2O2/DTPA, as
described under "Materials and Methods," followed by incubation
with 100 µM NBD for 30 min. All reagents were washed out
by filtration through Microcon CM-10 filters prior to absorbance
measurements. The Cys-S-NBD conjugate (solid line)
was generated by incubating denatured 20 S proteasome (40 µg) in
standard buffer with 100 µM NBD. Proteasome was denatured
by preincubation in 5 M guanidine solution buffered in 50 mM Tris/HCl, pH 7.5. Spectra were recorded with a Hitachi
spectrophotometer.
|
|
We also reacted denatured 20 S proteasome preparations with the -SH
reactant mBrB (22). Denatured 20 S proteasome samples were preincubated
with DTT or GSH. After incubation, DTT and GSH were removed, and the
samples were treated overnight with 150 µM mBrB.
Proteasome-Cys-bimane conjugates were detected by fluorescence emission
recorded after removal of mBrB. DTT-reduced samples showed fluorescence
emission at least twice as high as control samples, whereas GSH-treated
samples showed reduced fluorescence, probably because 20 S
proteasome mixed disulfides could not conjugate with mBrB (Fig.
4). This result indicates that GSH does
not play the role of a reducing compound as DTT, but probably GSH was
incorporated into the protein structure by
S-conjugation.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
mBrB incorporation into the 20 S core after
DTT and GSH preincubation. 20 S proteasome (10 µg) was incubated
in standard buffer for 30 min with DTT or for 10 min with GSH at room
temperature at the indicated concentrations. GSH or DTT was removed,
and protein samples were washed in standard buffer by filtration. The
Cys-mBrB conjugate was generated by overnight incubation with 150 µM mBrB at 4 °C in the dark. After incubation, 20 S
proteasome was precipitated with 20% trichloroacetic acid
followed by washing three times with 10% trichloroacetic acid. The
protein was dissolved in 5 M guanidine buffered in 0.1 M Tris/HCl, pH 7.5. Fluorescence emission was recorded at
476 nm (excitation at 400 nm). Results are expressed as means ± S.D. of three independent experiments. *, p < 0.000025. **, p at least 0.001 (ANOVA).
|
|
To demonstrate effectively that the effect of GSH on proteasomal
activity is because of S-glutathionylation, 20 S proteasome preparations were incubated with the GSH-biotin derivative according to
the protocol described in the literature (18). This method allows the
direct determination of protein S-glutathionylation, because
biotinylated GSH-proteasome complexes can be isolated by the
streptavidin affinity procedure. 20 S proteasome pretreatment with
H2O2 increased GSH incorporation (Fig.
5). The protein concentration determined
after protein elution from the streptavidin-agarose beads was 4-fold
higher in samples pretreated with H2O2. Protein recovered from control and H2O2-treated samples
after elution from the streptavidin beads was 10 and 40.5 µg,
respectively (the amount of protein reacted with GSH-biotin was the
same in both samples, i.e. 300 µg). This result is direct
proof that 20 S proteasome is susceptible to
S-glutathionylation by means of GSH addition to Cys-SOH.
Because a significant amount of S-glutathionylated 20 S
proteasome was detected in the control sample, part of its Cys residues
were probably already oxidized to Cys-SOH (Fig. 5). It should be
emphasized that the reaction with GSH-biotin was performed under
non-denaturing conditions. The preparation was brought to denaturing
conditions only after incubation with and removal of GSH-biotin to
displace the 20 S core from the streptavidin-agarose beads. Thus the
results described in Fig. 5 do not allow us to predict how many or
which subunits of the 20 S core were S-glutathionylated. We
tried to denature the proteasome just after incubation with GSH-biotin
and prior to the streptavidin incubation to determine approximately how
many and which subunits would be S-glutathionylated. However, under these conditions, the proteasome concentration of
control and H2O2-treated samples recovered from
the agarose beads was not sufficient to be detected on the gel by
Coomassie Blue staining. This is an indication that perhaps very few or even only one subunit might be S-glutathionylated.

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 5.
20 S proteasome
S-glutathionylation by GSH-biotin.
Glutathionylated 20 S proteasome was purified from control (300 µg)
and H2O2-treated (300 µg) samples after
incubation with GSH-biotin followed by SDS-PAGE. The complete
experimental procedure is described under "Materials and Methods."
The gel shown is representative of three independent
experiments. The left lane refers to 5 µg of a standard
preparation of 20 S proteasome, and the middle and
right lanes refer to the GSH-biotin-incubated control (10 µg) and to H2O2-treated samples (40.5 µg),
respectively.
|
|
Comparative Catalytic Regulation by GSH among 20 S Proteasome
Structures--
Chymotrypsin-like activity of 20 S proteasome from
Methanosarcina thermophila, an Archaeon bacterium, was
assayed in the presence of GSH and NEM for comparison to its
counterpart, the yeast 20 S proteasome. The Archaeon proteasome was not
affected by GSH and was inhibited slightly by NEM (Table
II). In the case of mammalian 20 S
proteasome, both GSH and GSSG at micromolar concentrations activated the chymotrypsin-like activity, whereas it was inhibited by
millimolar concentrations (15). Moreover, 20 S mammalian proteasomal
activity was inhibited strongly by NEM. On the other hand, the yeast 20 S core is inhibited only by reduced GSH (Fig. 1) and is inhibited
strongly by NEM (Table I) whereas the Archaeon proteasome is not
affected by any GSH form and is only affected slightly by NEM (Table
II).
View this table:
[in this window]
[in a new window]
|
Table II
Effect of Cys modification on Archaeon proteasome activity
Archaeon proteasome was purchased from ICN Biomedical Research
Products. 25-50 µg/ml was preincubated for 10 or 30 min at room
temperature in the presence of GSH or NEM, respectively, at the
indicated concentrations, followed by a further 1-h incubation at
37 °C with 10 µM s-LLVY-MCA. Hydrolysis determination
was described under "Materials and Methods." Results are means ± S.D. of three independent experiments.
|
|
These results indicate a possible role of Cys residues inside the 20 S
core controlling its catalytic activity. Comparing Cys residue content
among archaeon, yeast, and mammalian (human) 20 S proteasomes, 28, 74, and 100 (108 total Cys) reduced Cys residues are found, respectively.
Most likely the differential effect of GSH addition on proteasome
activity is related to non-conserved Cys residues. Our belief is that
the 20 S proteasome core has evolved as an important sensor of cellular
redox status by modification in its catalytic activity brought about by
changes in its Cys residues, promoting the cellular response to redox alterations.
In Vivo 20 S Proteasome Glutathionylation--
Our next approach
was to search for in vivo 20 S proteasome
S-glutathionylation. Our hypothesis was that proteasome
would be sensitive to intracellular reductive capacity, being more
active as the cell reductive environment is maximized. This might be an
additional way for cells to trigger the signaling response to oxidative
alterations. To test this hypothesis, we treated cells with increasing
H2O2 concentrations. After treatment, the hydrolysis of the fluorogenic substrate s-LLVY-MCA was determined in
preparations of 20 S proteasome isolated from the cell extract with the
anti-FLAG system and in the proteasome-free cell extract obtained after
20 S proteasome extraction. The 20 S core-anti-FLAG antibody complex
bound to agarose beads was assayed for the hydrolysis of the
fluorogenic peptide s-LLVY-MCA before and after incubation in the
presence of 20 mM NaBH4 to reduce the S-S
bridges formed by mixed disulfides or to reduce Cys-SOH to Cys-SH.
NaBH4 can also reduce carbonyl groups or Schiff bases
formed by the oxidation of some specific amino acid residues to alcohol
derivatives (23). Our purpose in using NaBH4 was to reduce
oxidized Cys residues inside the 20 S core, thereby recovering
proteasome sulfhydryls and promoting GSH release from the core. GSH was
assayed in the supernatant recovered after reduction with
NaBH4, as described under "Materials and Methods."
The reductive cellular capacity was evaluated by the determination of
the GSH/GSSG ratio. We also determined cell viability and the formation
of carbonyl protein as a marker of oxidative stress (Table
III).
View this table:
[in this window]
[in a new window]
|
Table III
Proteasomal activity and redox parameters upon H2O2
cell treatment
Cells were grown in YPD medium to A600 of 0.6-0.8
followed by a further 1-h incubation with H2O2 added to
the medium at the final concentrations shown. After incubation,
aliquots were taken for cell viability assay. The remaining cells were
harvested by centrifugation and washed twice with water. Cell extract
preparation, 20 S proteasome purification by immunoprecipitation,
reduction with NaBH4, and measurements of proteolysis, total
and oxidized glutathione, GSH released from 20 S proteasome, and
carbonyl proteins are described under "Materials and Methods." ND,
not determined.
|
|
H2O2 treatment leads to a significant increase
in the formation of protein carbonyl when added to the culture medium
at a concentration of at least 0.5 mM (Table III). At this
concentration, H2O2-promoted oxidative stress
was accompanied by decreased reductive capacity of the cell, as
evaluated by the GSH/GSSG ratio, which dropped to 68.5% compared with
that found in control cells. Under the same conditions, proteasomal
activity decreased 50% before incubation with NaBH4 and
was partially recovered after reduction with NaBH4 to 70%
of control samples (Table III). Hydrolysis of the fluorogenic peptide
s-LLVY-MCA, determined in the proteasome-free extract, increased by
60%. Reports in the literature (24-26) show that mammalian cells
under oxidative stress show increased proteolysis. The cited authors
assume that increased proteolysis is because of increased proteasomal
activity such as that seen in vitro when oxidized substrates
show increased hydrolysis levels by purified 20 S proteasome
preparations from mammalian cells (3). However, our results show that
yeast 20 S proteasome is highly affected by oxidative stress whereas
hydrolysis not dependent on proteasome is increased (Table III).
The reason for the partial recovery of proteasome activity after
NaBH4 treatment (Table III) is probably the reduction of
Cys-SOH or Cys-SSG to Cys-SH. However, other oxidative processes are
responsive to reduction by NaBH4, e.g. formation
of carbonyl and Schiff bases (19), but in this case the original amino
acid structures are not regenerated as expected for Cys-SOH and
Cys-SSG. GSH release from the 20 S core after reduction with
NaBH4 (Table III) is strong evidence that proteasomal
activity might be regulated by S-glutathionylation under
intracellular oxidative conditions, and consequently decreased reductive capacity, here attested by a reduced GSH/GSSG ratio upon
H2O2 cell treatment.
Taken together, results obtained in vivo after cell
treatment with H2O2 indicate that loss of
reductive cellular capacity, according to GSH/GSSG ratios and protein
carbonyl levels, is associated with loss of 20 S proteasomal activity
and with its S-glutathionylation. These data are an
indication of redox modulation of 20 S proteasome activity.
 |
DISCUSSION |
It is becoming increasingly apparent that many oxidant-sensitive
proteins are S-glutathionylated in response to intracellular redox status (6, 18, 27) (reviewed in Ref. 28). Cys-SOH is an
intermediate form involved in redox regulation and catalysis by protein
sulfhydryl groups (reviewed in 6). Distinct fates for protein Cys-SOH
have been considered (11, 14) whereby S-glutathionylation would be one of the mechanisms modulating protein activity. Protein S-glutathionylation is a reversible process, and there is
considerable evidence that GSH release is controlled enzymatically,
probably by glutaredoxin (29).
In this manuscript we describe the mechanism by which 20 S
proteasome is glutathionylated in vitro. Taken together,
those results indicate that during moderate oxidative stress Cys
residues inside the 20 S proteasome core might be oxidized to Cys-SOH, reversibly protected by S-glutathionylation, and most likely
deglutathionylated after cellular recovery from oxidative stress.
From the data in Table III we calculated that one to two molar GSH was
released per mol of 20 S proteasome, considering that the molecular
mass of the yeast protease is ~700 kDa. GSH was not detected
in 20 S proteasome samples obtained from control cells, probably
because of the sensitivity of the assay utilized for GSH detection. The
low GSH concentration released from the 20 S core is a suggestion that
S-glutathionylation of 20 S proteasome at the physiological
level is highly specific; probably very few Cys residues in the core
are prone to S-glutathionylation during metabolic processes.
Our hypothesis is that S-glutathionylation of the 20 S
proteasome is coupled to the oxidation of sulfhydryls, according to the
reaction shown above.
Comparing the results described here about the effect of GSH and GSSG
on proteasomal activity from yeast (see Figs. 1 and 2) and M. thermophila (Table II) to earlier results (15) obtained for
mammalian 20 S proteasome, it is clear that the 20 S proteasome homologues respond differentially to changes in redox conditions. In
this regard, it is important to observe that the mammalian counterpart
has either long- or short-lived proteins as substrates, and its
localization inside cells is widespread; it is found in the cytoplasm
and nucleus (30). On the other hand, the yeast proteasome is associated
with the degradation of short-lived proteins and localized mainly
inside the cell nucleus, and perhaps not more than 20% occurs in the
cytoplasm associated with the nuclear endoplasm reticulum network
(31).
It is interesting to point out that GSH distribution inside yeast cells
is still not clear. Although it has been demonstrated already that GSH
is distributed inside the mammalian nucleus at concentrations as high
as in the cytoplasm (32), its presence inside the yeast nucleus is not
clear, neither is the distribution of glutarredoxin isoforms
inside yeast cells clear thus far. A mammalian nuclear isoform was
described already (33, 34) whereas information on yeast nuclear
isoforms is still lacking. Because glutaredoxin activity is coupled to
direct GSH consumption, its presence inside the nucleus would be strong
evidence of GSH distribution in the nucleus. Considering proteasome
distribution and its role in the yeast cell, as discussed above,
elucidation of GSH distribution inside yeast cells is an important
matter to corroborate the findings discussed here.
Another important difference suggested in the literature (30, 31) is
that yeast proteasome is always capped by the 19 S regulatory unit in
contrast to the finding that its counterpart, the mammalian 20 S
proteasome, is found in 3-4-fold excess over the 19 S regulatory unit.
In our opinion, even if it is true that the yeast proteasome is always
capped with the 19 S regulator, this does not rule out the possibility
that the catalytic unit represented by 20 S proteasome is regulated
independently of either protein ubiquitinylation or substrate
recognition by the 26 S proteasome. Our results indicated that redox
regulation by glutathionylation is important in vivo (Table
III). Other examples of redox regulation and
S-glutathionylation have been described for other metabolic processes (27, 35, 36). Our results lead us to speculate that 20 S
proteasomal activity can be modulated by S-glutathionylation through the increased presence of oxidants, which would be used in
signaling processes. Besides, transient 20 S proteasome inhibition would decrease the hydrolysis of proteins responsible for redox signaling, e.g. AP-1-like factors. AP-1-like proteins are
sensors of the redox state of the cell (37) and, as already
demonstrated in mammalian cells, are degraded by the proteasome (38).
The metabolic advantage of protein S-glutathionylation in
redox signaling is the prevention of irreversible oxidation of the Cys
thiol group to Cys-SO2H or Cys-SO3H, permitting
protein reactivation by reduction. Such mechanism might work in
parallel to the main mechanism controlling proteasome-mediated
proteolysis, i.e. ubiquitinylation. The relationship between
proteasome glutathionylation and ubiquitinylation of substrates remains
to be established.