From the Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Instituto Reina Sofía de Investigaciones Nefrológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-28006 Madrid, Spain
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study addresses potential molecular
mechanisms underlying the inhibition of the transcription factor c-Jun
by nitric oxide. We show that in the presence of the physiological
sulfhydryl glutathione nitric oxide modifies the two cysteine residues
contained in the DNA binding module of c-Jun in a selective and
distinct way. Although nitric oxide induced the formation of an
intermolecular disulfide bridge between cysteine residues in the
leucine zipper site of c-Jun monomers, this same radical directed the
covalent incorporation of stoichiometric amounts of glutathione to a
single conserved cysteine residue in the DNA-binding site of the
protein. We found that covalent dimerization of c-Jun apparently did
not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of
transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced
S-glutathionylation and inhibition of c-Jun involves the
formation of S-nitrosoglutathione. In conclusion, our
results support the reversible formation of a mixed disulfide between
glutathione and c-Jun as a potential mechanism by which nitrosative
stress may be transduced into a functional response at the level of transcription.
The free radical nitric oxide
(NO)1 has emerged as a major
signaling molecule in the immune, cardiovascular, and nervous system (1-5). Accumulating evidence suggests that NO may play a role in the
redox control of transcription by modulating the DNA binding activity
of transcriptional activators such as OxyR (6), nuclear factor- A recent study with purified c-Jun and c-Fos, which constitute the
transcriptional activator AP-1, indicates that NO inhibits the DNA
binding activity of AP-1 by modifying cysteine residues in the
DNA-binding site of these proteins through as yet unknown mechanisms
(12). These findings fit well with previous studies on truncated Fos
and Jun constructs which mapped redox regulation of AP-1 to a single
conserved cysteine residue located in the basic DNA-binding site of
c-Fos and c-Jun (13, 14). Reduction of this critical cysteine residue
by chemical-reducing agents such as DTT and 2-mercaptoethanol or by the
DNA repair enzyme Ref-1 has been shown to convert the inactive and
presumably oxidized form of c-Fos and c-Jun into an active state that
is permissive for DNA binding (13, 15). In vitro, oxidation
of c-Jun and concomitant inhibition of its DNA binding activity occur
rapidly when the concentration of the reducing agent in the incubation medium (e.g. DTT or 2-mercaptoethanol) falls below 0.2 mM (16). The conclusion that NO inhibits AP-1 DNA binding
by specifically reacting with cysteine residues in c-Jun and c-Fos,
however, was reached from the observation that NO concentrations >0.1
mM inactivate the transcription factor in the presence of
low concentrations (0.1 mM) of the dithiol DTT (12). Given
the high capacity of NO to decrease thiol levels by
S-nitrosylation and oxidation (17), this raises the question
if the observed effects of NO on Jun/Fos DNA binding are in fact
directly related to a protein modification by NO such as
S-nitrosylation or can be attributed to the oxidation of the
transcription factor as described by Curran and co-workers (13, 16) as
a consequence of NO-induced thiol depletion.
To address this issue, we analyzed purified recombinant c-Jun DNA
binding domains for NO-induced thiol modifications and concomitant changes in DNA binding activity. In our in vitro system,
special emphasis was given to the role of the reduced sulfhydryl
compound GSH, which is present in concentrations of 1-10
mM in mammalian cells (18). GSH not only protects
oxidant-sensitive protein thiols against oxidative damage (19, 20) but
also critically determines the biological activity of NO (21-23). We
show here that in the presence of physiologically relevant
concentrations of GSH, NO inhibits c-Jun DNA binding in
vitro by specifically targeting the formation of a mixed disulfide
with GSH to a conserved cysteine residue in the DNA-binding site of the
transcription factor. Furthermore, we provide experimental evidence
that GSNO, which is formed by the reaction of NO with GSH, may mediate
the NO-dependent S-glutathionylation of
c-Jun.
Materials--
GSH (free acid, SigmaUltra) and GSSG (free acid,
SigmaUltra) were purchased from Sigma and Aldrich. DEA/NO and GSNO were
from Alexis Biochemicals. Yeast glutathione reductase (120 units/mg) was provided by Roche Molecular Biochemicals. Stock solutions of
[3H]GSH were prepared at a final concentration of 20 mM by the addition of 10 volumes of a freshly prepared
solution of 22 mM unlabeled GSH (free acid, SigmaUltra) in
H2O to 1 volume of tritium-labeled glutathione
([3H]GSH, 45-50 Ci/mmol, ~0.02 mM, NEN
Life Science Products) and stored in small aliquots at Preparation of Wild Type and Mutant c-Jun DNA Binding
Domains--
The insert coding for the DNA binding domain of human
c-Jun, corresponding to amino acids 223-327 of the translated sequence with the GenBankTM accession number J04111, was amplified
by polymerase chain reaction and cloned into the
BamHI-HindIII site of the expression vector
pQE-30 (Qiagen). The obtained hexahistidine fusion protein, which
encodes for one cysteine in the basic DNA-binding site (amino acid 269)
and a second cysteine in the leucine zipper (amino acid 320) of c-Jun,
was designated as CC-Jun. Cysteine 269 to serine (SC-Jun) and cysteine
320 to serine (CS-Jun) mutants were generated by polymerase chain
reaction-directed mutagenesis and cloned into the
BamHI-HindIII site of the expression vector
pQE-30. The obtained c-Jun plasmids were transformed into competent
Escherichia coli (M15[pRep4], Qiagen) according to the
instructions of the manufacturer. Recombinant clones were verified by
restriction analysis and dideoxynucleotide sequencing. The recombinant
proteins were expressed and purified by nickel-chelate chromatography
as described (16). The obtained protein preparation was dialyzed
excessively against a 25 mM phosphate buffer (pH 7.4),
containing 1 mM EDTA, 5% (v/v) glycerol, 0.1% (v/v)
2-mercaptoethanol, 0.01% (v/v) Nonidet P-40, and concentrated up to
~0.5 mM using Vivapore 20 concentrators (Vivascience).
Protein concentrations were determined by amino acid analysis. Purity of the proteins was estimated to be >95% as judged by
Coomassie-stained SDS gels.
Detection of Covalently Linked c-Jun Homodimers--
c-Jun DNA
binding domains (10 µM) were incubated for 30 min at
37 °C in a 20 mM Tris/HCl buffer (pH 7.5), containing 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5% (v/v) glycerol, 0.01% (v/v) Nonidet P-40
(buffer A), and 3 mM GSH in the absence and presence of
DEA/NO or GSNO. Reactions were stopped by the addition of iodoacetamide (50 mM) and incubation for further 30 min at room
temperature. Samples (4 µg of protein) were subjected to non-reducing
SDS-PAGE on discontinuous Tris/glycine slab gels (7 × 8 cm),
which contained acrylamide and bisacrylamide at final concentrations of
16 and 0.1% (w/v), respectively (25). Gels were stained for protein with Coomassie Blue R-250 and analyzed by densitometry.
Detection of a Mixed Disulfide between c-Jun and
Glutathione--
c-Jun DNA binding domains (10 µM) were
incubated in a final volume of 0.1 ml at 37 °C in buffer A
containing 3 mM [3H]GSH (~3 × 106 cpm) in the absence and presence of DEA/NO or GSNO. For
some experiments, incubations were performed in the presence of
[3H]GSNO, which was prepared from [3H]GSH
by nitrosation with acidified nitrite (26), or in the presence of 1 mM NADPH and 0.6 units of yeast glutathione reductase. To
isolate the glutathionylated protein by trichloroacetic acid precipitation, reactions were stopped by the addition of 0.9 ml of 10%
(w/v) ice-cold trichloroacetic acid and incubation on ice for 30 min.
Samples were centrifuged for 10 min at 20,000 × g, and
the supernatant was discarded. Subsequent to washing the precipitated protein three times with 0.9 ml of 10% (w/v) ice-cold trichloroacetic acid, the protein pellet was dissolved by treatment with 0.1 ml of 0.5 N NaOH for 20 min at 70 °C and assayed for incorporation of [3H]GSH by liquid scintillation counting. Results were
corrected for protein recovery (68 ± 7%, mean ± S.E.,
n = 6) and blank values (
To study the reversibility of GSNO-induced c-Jun glutathionylation,
c-Jun DNA binding domains (10 µM CC-Jun) were incubated in a final volume of 0.8 ml for 1 h at 37 °C in buffer A
containing 3 mM [3H]GSH (~3 × 107 cpm per ml) and 1 mM GSNO prior to
isolation of the S-glutathionylated protein by
chromatography on Sephadex G-25 columns (NAP-10, Amersham Pharmacia
Biotech). Protein-containing fractions were pooled, and stoichiometric
(0.8-1.1 mol of [3H]GSH per mol of protein)
incorporation of [3H]GSH was verified by trichloroacetic
acid precipitation as described above. To study dethiolation of c-Jun,
the [3H]GSH-labeled protein was incubated in a final
volume of 0.1 ml at 37 °C in buffer A in the absence and presence of
3 mM [3H]GSH (~3 × 107
cpm per ml) or 10 mM DTT. After 1 h, samples were
analyzed for [3H]GSH incorporation as described above.
GSH-induced dethiolation was expressed in percent of the radioactivity
that was released from the [3H]GSH-labeled protein by 10 mM DTT.
Determination of c-Jun DNA Binding Activity--
c-Jun DNA
binding domains (10 µM) were preincubated for 30 min at
37 °C in buffer A, which contained 3 mM GSH, in the
absence and presence of DEA/NO or GSNO. For the determination of DNA
binding activity by EMSA, 2-µl aliquots of the preincubation mixture
were diluted into a final volume of 18 µl of buffer A, which
additionally contained 0.2 mg/ml bovine serum albumin, 0.1 mg/ml
poly(dI-dC), and where indicated 1 mM DTT. Finally, 2 µl
of the 32P-radiolabeled double-stranded AP-1
oligonucleotide (5'-GGG CTT GAT GAG TCA GCC GGA CC-3') were added, and
the samples were incubated for further 30 min prior to electrophoresis
at 200 V on pre-electrophoresed 6% non-denaturing polyacrylamide gels
with 22 mM Tris borate, 0.5 mM EDTA as running
buffer. Gels were dried, visualized by autoradiography, and analyzed by densitometry.
To investigate the reversibility of GSNO-induced c-Jun inactivation,
c-Jun DNA binding domains (10 µM CC-Jun) were incubated in a final volume of 0.8 ml for 1 h at 37 °C in buffer A
containing 3 mM GSH and 1 mM GSNO prior to
isolation of the S-glutathionylated protein by
chromatography on Sephadex G-25 columns. Protein-containing fractions
were pooled, and aliquots (2 µl) were incubated for 1 h at
37 °C in the absence or presence of 1 mM GSNO with
increasing concentrations of GSH or 1 mM DTT in a final
volume of 20 µl of buffer A containing 0.2 mg/ml bovine serum
albumin, 0.1 mg/ml poly(dI-dC), and the 32P-labeled
oligonucleotide. Samples were cooled to room temperature, subjected to
electrophoresis on non-denaturing gels, and the gels analyzed by
autoradiography as described above. DNA binding activity of CC-Jun was
quantified by densitometry and expressed as percent of maximal DNA
binding of the DTT-reactivated reduced protein which was determined in
the presence of 1 mM DTT.
Determination of Nitrite, GSNO, and GSSG
Concentrations--
Nitrite concentrations were determined
photometrically by the Griess reaction (27). GSNO concentrations were
calculated from the absorbance at 340 nm using an extinction
coefficient of 0.75 mM Data Evaluation--
Data are presented as mean values ± S.E. with the number (n) of experiments in parenthesis.
Concentration-response curves were fitted to the experimental data by
the Hill equation. Statistical analysis of data was performed by
Student's t test and linear regression analysis.
NO-induced Inhibition of c-Jun DNA Binding Activity Involves a
Conserved Cysteine Residue in the DNA-binding Site of the Transcription
Factor--
Incubations of wild type c-Jun DNA binding domains
(CC-Jun) in the presence of 3 mM GSH and increasing
concentrations of the NO donor DEA/NO resulted in a
concentration-dependent inhibition of DNA binding activity
of the protein (Fig. 1). Concentrations of 0.01, 0.1, 0.5, and 1 mM of the NO donor inhibited
CC-Jun DNA binding to 94 ± 4, 70 ± 8, 44 ± 3, and
13 ± 3% (n = 4-9) of untreated controls,
respectively. DNA binding activity was restored by DTT, suggesting that
the modification of a cysteine residue may be involved in the
NO-mediated inhibition of CC-Jun.
The homodimeric c-Jun DNA binding domain contains two pairs of cysteine
residues, one located in its basic DNA-binding site (Cys-269) and one
located in the leucine zipper-like subunit interface (Cys-320). To
assign the inhibitory effect of NO to one of these two pairs of
cysteine residues, we compared the effects of NO on the DNA binding
activity of wild type and mutant c-Jun constructs. As shown in Fig.
2, NO clearly inhibited DNA binding
activity of CC-Jun (87 ± 3% inhibition, n = 9),
whereas DNA binding of the mutant with a cysteine to serine mutation in
the DNA-binding site (SC-Jun) was not significantly affected (8 ± 10% inhibition, n = 5). On the other hand, mutation of
the cysteine in the leucine zipper domain of the c-Jun DNA binding
domain (CS-Jun) did not attenuate the inhibitory effect of DEA/NO
(76 ± 11% inhibition, n = 5). These data,
therefore, render it likely that NO-mediated inhibition of c-Jun DNA
binding involves the modification of a single cysteine residue
(Cys-269) in the DNA-binding site of the protein.
NO-induced Formation of an Intermolecular Disulfide Bridge between
c-Jun Monomers Is Not Involved in the Inhibition of the Transcription
Factor by NO--
The formation of disulfide bonds between subunits of
the AP-1 transcription factor, which is composed by Jun/Jun
homodimers or heterodimers between Jun and Fos proteins, was suggested
as one potential mechanism by which NO might inhibit AP-1 DNA binding activity (12). As shown in Fig. 3, NO
released from 1 mM of the NO donor DEA/NO in the presence
of 3 mM GSH in fact induced an SDS-resistant dimerization
of wild type c-Jun DNA-binding subunits (CC-Jun). Under these
conditions, >70% of CC-Jun were converted into covalently linked
dimers as determined by non-reducing SDS-PAGE. The reversibility of
this effect by DTT suggests the formation of an intermolecular
disulfide bridge between one or both of the two cysteines located in
the DNA binding module of c-Jun monomers. A comparison of cysteine to
serine mutants shows that SC-Jun but not CS-Jun was converted into
disulfide-linked dimers demonstrating that NO specifically targets
disulfide formation to cysteine 320 in the leucine zipper domain of
c-Jun. Since this cysteine residue is apparently not involved in the
NO-mediated loss of c-Jun function (see Fig. 2), these data make
intermolecular disulfide formation a highly unlikely mechanism for the
inhibition of the transcription factor by NO.
NO Induces the Formation of a Mixed Disulfide between c-Jun and
GSH--
An alternative mechanism by which protein thiols may
transduce oxidative stress into a post-translational modification and functional response is the formation of a mixed disulfide with GSH (30,
31). To investigate if this mechanism may be involved in the NO-induced
inhibition of c-Jun, we exposed purified c-Jun DNA binding domains to 1 mM of the NO donor DEA/NO in the presence of 3 mM 3H-labeled GSH, and we isolated the covalent
[3H]GSH-protein adduct by trichloroacetic acid
precipitation. As shown in Fig.
4A, DEA/NO induced a
time-dependent incorporation of the radiolabel with an
apparent half-time of ~5 min and a maximal incorporation of 0.8-0.9
mol of [3H]GSH per mol of CC-Jun (closed
symbols). Control incubations in the absence of DEA/NO (open
symbols) did not yield any significant amounts of protein bound
[3H]GSH. DTT-labile incorporation of the radiolabel (see
"Experimental Procedures") suggests binding of
[3H]GSH to the protein via a disulfide bond.
Fig. 4B shows the dependence of [3H]GSH
incorporation on the concentration of DEA/NO. At concentrations of
0.01, 0.1, 0.5, and 1 mM, DEA/NO induced binding of
0.05 ± 0.02, 0.28 ± 0.11, 0.62 ± 0.06, and 0.81 ± 0.08 mol of [3H]GSH (n = 3-6) per mol
of protein, respectively. Half-maximal mixed disulfide formation was
estimated to occur at DEA/NO concentrations of ~300 µM.
According to a recently published mathematical model (32), the steady
state concentration of NO under these conditions, i.e. 300 µM DEA/NO at 37 °C and pH 7.4, reaches a peak level of approximately 20 µM at ~0.6 min and drops exponentially
to submicromolar concentrations within 25 min.
As shown in Fig. 4C, GSH incorporation into CC-Jun was
almost completely abolished by a cysteine 269 to serine mutation
(0.13 ± 0.05 mol [3H]GSH/mol SC-Jun,
n = 5), whereas a cysteine 320 to serine mutation virtually did not affect the degree of S-thiolation
(0.87 ± 0.01 mol [3H]GSH/mol CS-Jun,
n = 3). These data, therefore, demonstrate that NO
specifically targets the formation of a mixed disulfide to a single
cysteine residue in the DNA-binding site of c-Jun (Cys-269). The
finding that this cysteine residue is involved in the NO-induced inhibition of CC-Jun (see Fig. 2) suggests that NO-induced
S-glutathionylation mediates the inhibitory effect of NO.
This was confirmed by linear regression analysis of data from DNA
binding (Fig. 1) and [3H]GSH incorporation (Fig.
4B) experiments, which yields a highly significant
(p = 0.01) inverse linear correlation between relative DNA binding activity of CC-Jun and DEA/NO-mediated
S-glutathionylation of the protein (intercept, 1.00; slope,
NO-induced c-Jun S-Glutathionylation Is Not Mediated by a Change in
the GSH/GSSG Ratio--
Various mechanisms have been suggested to
account for the formation of a mixed disulfide between GSH and protein
thiols (33). GSSG may directly S-thiolate proteins via a
thiol/disulfide exchange mechanism. Dependent on the protein examined
half-maximal glutathionylation was observed at GSH/GSSG ratios ranging
from 27 to 10 NO-induced c-Jun S-Glutathionylation May Be Mediated by
GSNO--
Recently, GSNO has been reported to induce the
S-glutathionylation of aldose reductase (38). Under aerobic
conditions, NO reacts with oxygen to yield the nitrosating species
N2O3. In the presence of an excess of GSH,
hydrolysis of N2O3 to nitrite is competitive
with the rate of its reaction with GSH to GSNO (35). Accordingly, we
found that in the presence of 3 mM GSH 1.08 ± 0.04 mol of nitrite (n = 16) and 0.47 ± 0.01 mol of
GSNO (n = 16) were formed per mol of DEA/NO during an
incubation period of 60 min at 37 °C (Table I). To investigate if
GSNO induces the formation of a mixed disulfide between GSH and c-Jun,
we incubated CC-Jun with increasing concentrations of GSNO in the
presence of 3 mM 3H-labeled GSH, and we
analyzed the protein for
S-[3H]glutathionylation (Fig.
6, open symbols). One
mM GSNO induced the incorporation of 0.98 ± 0.07 mol
of [3H]GSH/mol of CC-Jun (mean ± S.E.,
n = 3). From the concentration-response curve shown in
Fig. 6, we calculated a half-maximally active GSNO concentration of 160 µM. In agreement with a rapid exchange of NO between GSNO
and [3H]GSH, as it can be expected from the high
trans-nitrosation rate (~70 M
The biological activity of GSNO can be explained in part by the
release of NO from the nitrosothiol due to copper-catalyzed homolytic
cleavage (40). Alternatively, a direct nucleophilic attack of protein
thiols on the nitrosothiol, which does not require cleavage of GSNO,
has been suggested as a potential mechanism for GSNO-mediated protein
thiolation (38). To address this issue, we measured NO release from
GSNO under conditions that elicited quantitative
S-glutathionylation of CC-Jun by determining the accumulation of nitrite in the presence of 1 mM GSNO and 3 mM GSH during a 1-h incubation at 37 °C. Under these
conditions, nitrite accumulation was barely detectable (1.5 ± 0.1 µM, n = 3), indicating that GSNO does not
release NO in quantities that could explain the GSNO-induced
S-glutathionylation of c-Jun. Identical results were
obtained when CC-Jun (10-100 µM) was included in the
incubations. Furthermore, in accordance with a previous report on the
stability of GSNO in the presence of chelating agents such as EDTA and
millimolar concentrations of GSH (28), we did not observe any
significant decomposition of the nitrosothiol (<2% of total GSNO) as
determined by UV spectroscopy, i.e. by monitoring the
absorbance of the incubation mixture at 340 nm.
Inhibition of c-Jun by GSNO-induced S-Glutathionylation Is
Reversible--
To investigate if the GSNO-mediated inhibition of
c-Jun can be reversed by GSH, we S-glutathionylated CC-Jun
by incubation with GSNO and separated the protein from GSNO by
chromatography on Sephadex G-25 columns. Subsequently, the isolated
protein was incubated with increasing concentrations of GSH and
analyzed for DNA binding activity by EMSA (Fig.
7A, upper panel, and Fig.
7B, closed symbols). GSH concentrations of 0.03, 0.3, 3, and 10 mM induced a concentration-dependent
recovery of DNA binding activity to 11 ± 8, 39 ± 10, 79 ± 9, and 89 ± 4% (n = 4-6) of maximal
DNA binding activity (i.e. DNA binding activity of the
DTT-reactivated, reduced transcription factor), respectively. In the
presence of 1 mM GSNO (Fig. 7A, lower panel, and
Fig. 7B, open symbols) DNA binding activity was only
marginally increased by co-incubations with GSH at concentrations up to
3 mM ( NO and NO donor compounds such as S-nitrosothiols and
nitrosyl-iron complexes have been implicated in the redox control of protein function in terms of biological signaling as well as
nitrosative stress (41-43). Dependent on the reactivity and structural
context of the protein thiol on one hand and the reactivity of the
NO-derived species on the other hand, various mechanisms may account
for the post-translational modification of proteins by NO. It has been
suggested that NO may reversibly modify protein-bound cysteines by at
least four distinct mechanisms including the following: (i) the
covalent attachment of an NO moiety to the thiol, i.e. S-nitrosylation; (ii) the reversible oxidation to sulfenic
acid; (iii) the formation of intra- or intermolecular disulfide
bridges; and (iv) the formation of a mixed disulfide with GSH,
i.e. S-glutathionylation (44). Here we show that
NO and GSNO inhibit DNA binding activity of c-Jun by selectively
targeting the formation of a mixed disulfide with GSH to a conserved
cysteine residue in the basic DNA-binding site of the transcription
factor. A replot of data (Fig. 8) from densitometric analysis of DNA binding assays against
[3H]GSH incorporation into CC-Jun, which includes data
from assays performed with DEA/NO and GSNO (see Figs. 1, 4B,
and 6), shows a highly significant (p < 0.001) inverse
linear relationship between c-Jun S-glutathionylation and
relative DNA binding activity (intercept, 0.97; slope,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(7), and c-Myb (8) through S-nitrosylation of redox-sensitive thiols. Furthermore, NO has been reported to inhibit DNA binding of the transcription factor AP-1 in cerebellar granular cells (9) and to be involved in the post-transcriptional attenuation of
AP-1 during NO-induced neuronal cell death (10). Of interest, interferon
was shown to induce a down-regulation of AP-1 DNA binding activity in human brain-derived cells. This phenomenon is
associated with the development of neuroinflammatory diseases and was
found to be due to cytokine-mediated induction of NO synthase in these
cells (11). However, the molecular mechanisms underlying the inhibition
of AP-1 DNA binding by NO remain to be established.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
Throughout the text, this preparation of the radiolabeled thiol will be
referred to as [3H]GSH. As determined by high pressure
liquid chromatographic analysis (24) and in agreement with the
specifications provided by the manufacturer, the purity of
[3H]GSH was
98%. The only detectable contamination was
GSSG (
2%).
0.1 mol of radiolabeled GSH
per mol of protein), which were determined as non-DTT-releasable
radiolabel by treating the [3H]GSH-protein adduct for 60 min at 37 °C with 10 mM DTT prior to trichloroacetic
acid precipitation.
1 cm
1
(28). GSSG concentrations were determined by a coupled assay as
glutathione reductase-dependent oxidation of NADPH (29). Briefly, samples (0.1-0.7 ml) were assayed for GSSG in a final volume
of 1 ml of a 20 mM triethanolamine/HCl buffer (pH 7.6) containing 0.2 mM EDTA and 0.05-0.2 mM NADPH
by addition of 0.6 units of yeast glutathione reductase (120 units/ml)
and monitoring the absorbance decrease at 340 nm. NADPH consumption was
quantified using an extinction coefficient of 6.34 mM
1 cm
1. To study the effect of
a GSH-regenerating system on NO-induced oxidation of GSH to GSSG, GSH
(3 mM) was co-incubated with DEA/NO (1 mM) and
CC-Jun (10 µM) in 0.5 ml of buffer A for 1 h at
37 °C in the absence or presence of 6 units/ml glutathione reductase and 1 mM NADPH. Subsequently, the reductase was removed by
rapid filtration through microfilters (cut-off, 10 kDa), and aliquots of the filtrate were assayed for GSSG as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (72K):
[in a new window]
Fig. 1.
Inhibition of CC-Jun DNA binding by NO.
Wild type c-Jun DNA binding domains (CC-Jun) were incubated at final
concentrations of 10 µM for 30 min at 37 °C in a 20 mM Tris/HCl buffer (pH 7.5), containing 50 mM
NaCl, 5 mM MgCl2, 1 mM EDTA, 5%
(v/v) glycerol, 0.01% (v/v) Nonidet P-40, and 3 mM GSH in
the presence of increasing concentrations of DEA/NO. Aliquots (2 µl)
were assayed for DNA binding activity by EMSA in the absence and
presence of 1 mM DTT as described under "Experimental
Procedures." The shown autoradiograph is representative of four
experiments.
View larger version (91K):
[in a new window]
Fig. 2.
Involvement of cysteine residues in the
inhibition of CC-Jun DNA binding by NO. Wild type (CC-Jun) and
mutant c-Jun DNA binding domains, in which either the cysteine located
in the DNA-binding site (SC-Jun) or adjacent leucine zipper (CS-Jun)
were substituted by serine, were incubated with 1 mM DEA/NO
in the presence of 3 mM GSH and analyzed for DNA binding
activity by EMSA as described under "Experimental Procedures." The
shown autoradiograph is representative of five experiments.
View larger version (25K):
[in a new window]
Fig. 3.
NO-mediated formation of an intermolecular
disulfide bond between CC-Jun monomers. Wild type (CC-Jun) and
mutant c-Jun DNA binding domains, in which either the cysteine located
in the DNA-binding site (SC-Jun) or adjacent leucine zipper (CS-Jun)
were substituted by serine, were incubated for 30 min at 37 °C with
1 mM DEA/NO in the presence of 3 mM GSH and
analyzed for covalent dimerization by non-reducing SDS-PAGE as
described under "Experimental Procedures." The shown gels are
representative of at least four similar experiments.
View larger version (16K):
[in a new window]
Fig. 4.
NO induces the formation of a mixed disulfide
between glutathione and CC-Jun. Wild type c-Jun DNA binding
domains (10 µM CC-Jun) were incubated for the indicated
times at 37 °C in the buffer described in the legend to Fig. 1,
which contained 3 mM [3H]GSH, in the absence
(open symbols) or presence (closed symbols) of 1 mM DEA/NO (A). For DEA/NO concentration-response
curves (B), incubation times were 60 min. For the comparison
of wild type (CC-Jun) and mutant c-Jun proteins, in which either the
cysteine located in the DNA-binding site (SC-Jun) or adjacent leucine
zipper (CS-Jun) were substituted by serine, the time of incubation and
the final concentration of DEA/NO were 60 min and 1 mM,
respectively (C). Protein S-glutathionylation was
determined as trichloroacetic acid-precipitable, DTT-labile
[3H]GSH incorporation as described under "Experimental
Procedures." Data are mean values ± S.E. of three to five
experiments.
1.01; r = 0.99).
5 (34). NO was reported to oxidize GSH to
GSSG under anaerobic conditions, at low GSH/NO ratios, or via a
secondary reaction of GSNO with GSH (22, 35-37). In our experimental
system, GSSG concentrations were 15 ± 4 µM
(n = 6) under control conditions, i.e. in
the presence of 3 mM GSH and absence of an NO donor (Table I). During a 60-min incubation at
37 °C, DEA/NO at concentrations of up to 500 µM
oxidized less than 3% of the total amount of GSH (3 mM at
t = 0) to GSSG. At a concentration of 1 mM, DEA/NO
converted ~16% of GSH into GSSG, which results in a decrease of the
GSH/GSSG ratio to values <10. These data raise the possibility that an NO-induced shift in the GSH/GSSG ratio may mediate c-Jun mixed disulfide formation. To address this issue, we analyzed CC-Jun for
NO-induced [3H]GSH incorporation under conditions where
GSSG was recycled continuously to GSH by glutathione reductase (Fig.
5). In the presence of the GSH-regenerating system, NO-dependent GSSG formation was
almost completely suppressed (20 ± 11 µM,
n = 3) as compared with controls (241 ± 13 µM, n = 3). This ~10-fold decrease in
GSSG, which resulted in a >15-fold increase of the GSH/GSSG ratio from
<10 to ~130, did not significantly (p > 0.5) affect
mixed disulfide formation (0.76 ± 0.04, n = 3 versus 0.81 ± 0.08, n = 5). Thus,
these data argue against the involvement of GSSG in the NO-induced
S-glutathionylation of c-Jun.
Nitrite, GSSG, and GSNO formation in incubations of GSH with DEA/NO
View larger version (15K):
[in a new window]
Fig. 5.
Effect of a GSH-regenerating system on
NO-mediated GSSG formation and CC-Jun
S-glutathionylation. Wild type c-Jun DNA binding
domains (10 µM CC-Jun) were incubated with 1 mM DEA/NO in the buffer described in the legend to Fig. 1,
which contained 3 mM unlabeled (GSSG determinations) or
3H-labeled ([3H]GSH incorporation) GSH.
Incubations were performed for 60 min at 37 °C in the absence
(control) or presence (GSSG reductase) of 6 units/ml glutathione
reductase and 1 mM NADPH. Samples were assayed for GSSG
formation (closed bars) and [3H]GSH
incorporation into CC-Jun (open bars) as described under
"Experimental Procedures." Data are mean values ± S.E. of
three to five experiments.
1 × s
1) between equivalent thiol groups (39), essentially the
same results were obtained when unlabeled GSNO was replaced by
[3H]GSNO (not shown). GSNO-induced
S-glutathionylation of CC-Jun was paralleled by a loss of
DNA binding activity of the transcription factor (Fig. 6, closed
symbols) and mapped to Cys-269 (not shown). Linear regression
analysis confirmed a highly significant (p = 0.002)
inverse linear correlation between relative DNA binding activity of
CC-Jun and GSNO-mediated S-glutathionylation of the protein
(intercept, 0.96; slope,
0.88; r = 0.96).
View larger version (16K):
[in a new window]
Fig. 6.
Concentration-dependent
S-glutathionylation and inhibition of CC-Jun by
GSNO. Wild type c-Jun DNA binding domains (10 µM
CC-Jun) were incubated with increasing concentrations of GSNO for 60 min at 37 °C in the presence of 3 mM
3H-labeled ([3H]GSH incorporation) or
unlabeled (DNA binding assay) GSH and assayed for [3H]GSH
incorporation (open symbols) and DNA binding activity
(closed symbols) as described under "Experimental
Procedures." Relative DNA binding activity is expressed as the ratio
between the DNA binding activity in the presence and absence of GSNO.
Data are mean values ± S.E. of three to five experiments.
10% of the DTT-reactivated protein) and only
partially (27 ± 9%) recovered at the highest GSH concentration
(10 mM) investigated. These data, therefore, indicate that
inhibition of c-Jun by GSNO-induced mixed disulfide formation may be
reverted by the physiological sulfhydryl GSH provided that GSNO, which
appears to antagonize the reduction of the mixed disulfide by GSH, is
removed from the system. To substantiate that reactivation of
GSNO-inactivated c-Jun is associated with dethiolation of the protein,
we incubated isolated, S-3H-glutathionylated
CC-Jun (see "Experimental procedures") in the absence and presence
of 3 mM [3H]GSH or 10 mM DTT for
1 h at 37 °C and assayed the protein for [3H]GSH
incorporation. In the presence of 3 mM
[3H]GSH, 77 ± 4% (n = 3) of the
total DTT-releasable radioactivity were liberated from the protein.
When GSNO (1 mM) was additionally present, we did not
detect any significant dethiolation (data not shown). The good
correlation between recovery of DNA binding activity and dethiolation
in the presence of 3 mM GSH suggests that c-Jun
S-glutathionylation may be reversed by physiologically relevant concentrations of GSH and render dethiolation a likely mechanism of GSH-induced reactivation of the transcription factor.
View larger version (24K):
[in a new window]
Fig. 7.
Reactivation of GSNO-treated CC-Jun by
GSH. A, GSNO-inactivated CC-Jun, which was prepared by
incubation with 1 mM GSNO in the presence of 3 mM GSH and isolated as described under "Experimental
Procedures," was incubated in the absence (upper panel)
and presence (lower panel) of 1 mM GSNO with
increasing concentrations of GSH (30 µM to 10 mM) or DTT (1 mM) for 1 h at 37 °C and
analyzed for DNA binding activity by EMSA (see "Experimental
Procedures"). The shown autoradiographs are representative of four to
six similar experiments. B, densitometric analysis of the
autoradiographs shown in A. Data from experiments performed
in the absence (closed symbols) or presence (open
symbols) of GSNO are expressed as the percent of DNA binding
activity recovered under each condition relative to the
DTT-reactivated transcription factor (mean values ± S.E.,
n = 4-6).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.92;
r = 0.97). The excellent correlation between NO/GSNO-mediated GSH incorporation into the Jun protein on one hand and
the concomitant loss of DNA binding activity on the other hand as well
as the site specificity of both phenomena suggest that
S-glutathionylation in fact accounts for the inhibition of the transcription factor DNA binding activity by NO and GSNO.
View larger version (17K):
[in a new window]
Fig. 8.
S-Glutathionylation of CC-Jun
correlates with a loss of its DNA binding activity. To correlate
c-Jun inhibition with S-glutathionylation, densitometric
data from DNA binding assays performed in the absence and presence of
DEA/NO and GSNO (Figs. 1 and 6) were re-plotted against the DEA/NO- and
GSNO-induced incorporation of [3H]GSH into CC-Jun (Figs.
4B and 6). Data, which are mean values ± S.E. of 3-9
experiments, were analyzed by linear regression (intercept, 0.97;
slope, 0.92; r = 0.97, p < 0.001).
Possible mechanisms of NO/GSNO-induced protein S-glutathionylation include the activation of the protein cysteine by S-nitrosylation or oxidation to a sulfenate which may subsequently react with GSH to the corresponding mixed disulfide (44, 45). Although we did not observe any S-nitrosylation of the relevant c-Jun cysteine as judged by UV/Vis spectroscopy of the DEA/NO-treated protein (not shown), we cannot exclude that S-nitrosylated c-Jun may be formed as a short lived intermediate. A c-Jun sulfenate, which has been proposed in a previous study to account for the redox regulation of AP-1 (13), may be another reactive intermediate in the modification of c-Jun by NO donors. In support of this hypothesis, we found that the spectroscopic sulfhydryl/sulfenate probe 7-chloro-4-nitro-2-oxa-1,3-diazole reacts with an intermediate, which was formed during the oxidation of CC-Jun by NO, to an adduct with an absorbance maximum at 350 nm. Although the formation of a compound with these spectral characteristics would be consistent with the intermediary conversion of a protein thiol to a sulfenate (46), quantitative analysis of these data revealed that less than 5% of the protein were scavenged as c-Jun sulfenate.2 Moreover, we did not detect any c-Jun sulfenate when GSNO was used instead of NO, which argues against a role for this intermediate in the NO/GSNO-mediated thiolation of c-Jun. Definitive conclusions about the formation of presumably short lived intermediates preceding NO/GSNO-dependent S-glutathionylation of c-Jun, however, await detailed kinetic analysis by stopped flow techniques.
An alternative explanation for the NO-induced S-glutathionylation of c-Jun may be the formation of GSNO due to S-nitrosylation of GSH by NO and subsequent reaction of GSNO with the protein. In keeping with a role for GSNO in the modification of c-Jun by NO, we found that the reaction of NO with GSH yields GSNO in concentrations (see Table I), which fitted well with the efficacy and potency of DEA/NO and GSNO to elicit both c-Jun S-glutathionylation and inhibition (see Figs. 4B and 6). The apparent lack of NO release from GSNO under our experimental conditions further supports the view that GSNO itself and not NO may be the reactive species involved in the transfer of the glutathionyl moiety to the c-Jun protein. This hypothesis fits well with a recent report showing that GSNO directly S-glutathionylates human aldose reductase via a nucleophilic attack of the enzyme thiol on the sulfur of GSNO (38). However, although apparently only a small portion of GSNO (<2%) decomposed under our experimental conditions, we cannot exclude the involvement of other as yet unidentified reactive species which might efficiently thiolate the protein at low concentrations. There is evidence that GSH and GSNO may react to the corresponding N-hydroxysulfenamide and, depending on the availability of GSH and oxygen, this adduct may undergo a number of reactions yielding GSSG, GSH sulfinic acid, GSH sulfenamide, GSH sulfinamide, GSH sulfenylhydroperoxide, and various presumably short lived radical species on the one hand and nitrite, N2O, and NH3 on the other hand (22). Thus, given the complex and as yet not entirely elucidated chemistry of the GSH/GSNO system, further studies are required to establish the molecular mechanism underlying GSNO-induced mixed disulfide formation.
We show that inhibition of c-Jun by NO and GSNO maps to a conserved cysteine residue in the DNA-binding site of c-Jun. This oxidant-sensitive cysteine residue has been identified in previous studies as the amino acid residue that provides redox sensitivity to c-Jun presumably by suffering reversible oxidation to a sulfenic acid (13, 14, 47). In support of a redox-dependent regulation of c-Jun DNA binding, in vitro as well as cell culture studies showed that depletion of reducing thiols in the incubation medium (16), treatment of AP-1 with GSSG or oxidized thioredoxin (48), depletion of intracellular GSH pools (48), or immunodepletion of the nuclear redox protein Ref-1 (15) attenuate AP-1 DNA binding activity. In contrast with the oxidative inactivation of c-Jun and c-Fos seen previously by Curran and co-workers (13) inhibition of c-Jun by NO/GSNO-induced S-glutathionylation occurs under reductive conditions, i.e. in the presence of millimolar concentrations of GSH and GSH/GSSG ratios >100 (see Table I and Fig. 5). Although the extension of c-Jun S-glutathionylation to in vivo systems remains to be established, these data raise the possibility that regulation of AP-1 DNA binding activity by oxidative and nitrosative stress may operate independently from each other.
GSNO-induced inhibition of c-Jun by S-glutathionylation appears to be a reversible process. In the presence of physiologically relevant concentrations of GSH, removal of the nitrosothiol resulted in dethiolation of c-Jun and recovery of its DNA binding activity. Given that GSNO may accumulate in situations of nitrosative stress on the one hand and may be subjected to decomposition via nonenzymatic copper-dependent mechanisms (28) or recently discovered nitrosothiol-metabolizing enzymes (49) on the other hand, these data suggest that reversible GSNO-dependent thiolation of c-Jun may be a control mechanism linking GSNO formation to regulation of transcription. Of note, the thioredoxin/thioredoxin reductase system, which has been implicated in the redox regulation of the AP-1 transcription factor (15, 50, 51), has been shown recently to cleave GSNO (52), to reverse inhibition of AP-1 by NO (12), and to reactivate RNA-binding proteins after exposure to NO (53). It remains to be investigated, however, if this nuclear redox system plays a role in the regulation of AP-1 by GSNO.
It is well established that reactive oxygen and nitrogen species play a
key role in the redox regulation of cellular activation, transcription,
proliferation, and cell death (44, 54-58). Within this concept of
oxidative and nitrosative stress, reversible
S-glutathionylation of oxidant-sensitive cysteines has been
established as one of the post-translational protein modifications that
may regulate protein function in response to oxidative stress or
protect proteins against irreversible oxidative damage (30, 31). It has
been shown that the activation of protein thiols by reactive oxygen species such as superoxide and hydrogen peroxide facilitates the formation of a mixed disulfide between protein thiols and glutathione (59). The results of this study, demonstrating reversible
GSNO-dependent S-glutathionylation of c-Jun
in vitro, suggest that S-thiolation of a
transcription factor triggered by reactive nitrogen species and
nitrosothiols may add a novel molecular mechanism to the concept of
nitrosative stress. In support of a potential role for mixed disulfide
formation as a signal by which nitrosative stress is sensed by cells,
S-thiolation of endothelial cell proteins in response to NO
has been reported recently (60). Given the striking structural
similarities between the positively charged DNA-binding site of c-Jun
and the DNA binding domain of a number of cysteine-containing transcription factors, including members of the Fos, ATF/CREB, Myb, and
Rel/NF-B families, NO-induced S-glutathionylation of a
basic DNA binding motif as exemplified by c-Jun may represent a general
mechanism by which nitrosative stress is transduced into a functional
response at the transcriptional level.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Fernando J. Corrales from the Department of Internal Medicine, Facultad de Medicina, Universidad de Navarra, Pamplona, Spain, for high pressure liquid chromatography analysis of GSH preparations. We thank Dr. Javier Rey (Centro de Investigaciones Biológicas, CSIC, Madrid, Spain) for the generous gift of a c-Jun plasmid and for critically reading the manuscript. We also thank Dr. Guillermo Giménez Gallego (Centro de Investigaciones Biológicas, CSIC, Madrid, Spain), Dr. José Antonio Bárcena (Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Córdoba, Spain), and Dr. Maria del Pilar Ramos Alvarez, Facultad de Ciencias Experimentales y Técnicas, Universidad San Pablo CEU, Boadilla del Monte, Spain) for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Biomed-2 grants from the European Community, Marie Curie Fellowship BMH4-CT98-5052 (to P. K.), Concerted Action Grant BMH4-CT96-0979 (to S. L.), Grant SAF 97-0035 from the Comisión Interministerial de Cienca y Tecnología (to S. L.), and a postgraduate fellowship of the Spanish Ministry of Education and Culture (to E. P. 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.
Both authors have contributed equally to this work.
§ To whom correspondence should be addressed: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Calle Velázquez 144, 28006, Madrid, Spain. Tel.: 34-91-561-1800-4419; Fax: 34-91-562-7518; E-mail: slamas{at}cib.csic.es (for S. L.) or pklatt{at}cib.csic.es (for P. K.).
2 P. Klatt, E. Pineda Molina, and S. Lamas, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NO, nitric oxide; DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt; GSNO, S-nitrosoglutathione; DTT, dithiothreitol, AP-1, activator protein-1; CC-Jun, wild type human c-Jun DNA binding domain; SC-Jun, human c-Jun DNA binding domain with a cysteine 269 to serine mutation; CS-Jun, human c-Jun DNA binding domain with a cysteine 320 to serine mutation; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|