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INTRODUCTION |
Iron regulatory proteins (IRP1 and
IRP2)1 are
trans-regulators that post-transcriptionally control the
expression of key proteins of iron metabolism by binding to hairpin
loop structures named iron-responsive elements (IREs) on their mRNA
(1-3). IRP binding to IREs in the 5'-untranslated region of ferritin
and erythroid aminolevulinate synthase results in inhibition of
translation (4-8), whereas binding of IRPs to multiple IREs in the
3'-untranslated region of transferrin receptor mRNA confers
stability against endonucleolytic degradation (9, 10).
IRP1/IRE binding activity is regulated by cellular iron levels. In
iron-depleted cells, high affinity of IRP1 for IREs diminishes iron
storage in ferritin and induces transferrin receptor synthesis, thereby
enhancing iron uptake via receptor-mediated endocytosis of serum
transferrin. Conversely, in iron-loaded cells IRP1 fails to bind RNA
but functions as an aconitase, converting citrate into isocitrate in
the cytosol (1, 2, 11). IRP1 is thus a bifunctional protein whose
activities appear to depend on the presence or absence of an intact
[4Fe-4S] cluster (12) which is ligated by three cysteine residues at
the active site (13, 14). The switch of IRP1 from aconitase to
IRE-binding protein has been explained by the removal of the Fe-S
cluster (2, 12, 13), but the mechanisms that underlie the insertion and
extrusion of the cluster need to be delineated. The second IRP
discovered, IRP2, shares 61% amino acid identity with IRP1, differing
by the insertion of a 73-amino acid sequence rich in cysteine residues (15, 16). IRP2 is also regulated by iron, but unlike IRP1, it is
rapidly degraded in cells that are iron-replete. Although IRP2 contains
the three conserved cysteine ligands for the [4Fe-4S] cluster of
IRP1, it seems not to have such a cluster, and it lacks aconitase
activity (17-19).
It is now clear that signals other than iron can regulate IRP1 and IRP2
activities and thus modulate cellular iron metabolism. Indeed, previous
studies showed that biosynthesis of NO, oxidative stress, and
phosphorylation increase IRP1/IRE binding in different cell types
(20-26). As regards stimulation by NO, previous results from our
laboratory showed that IRE binding by IRP1 is almost maximal when NO
synthase 2 is induced in macrophages by stimulation with both
interferon-
and lipopolysaccharide (20). The conversion of IRP1 from
holo- to apoprotein can also be elicited in vitro by
exposing cell cytoplasmic extracts to NO-releasing chemicals. However,
despite high concentrations of NO donor, we consistently observed that
IRP1 activation under these conditions was far from that observed in
intact cells (27). Moreover, exposure of purified recombinant IRP1 to
NO donors or to NO gas resulted in an even weaker IRP1 activation (20).
It seems therefore that simplification of the experimental system
results in the loss of a cellular component that plays a part in
NO-mediated IRP1 activation. Moreover, it is also worth recalling that
IRP1 and IRP2 are redox-sensitive proteins. Indeed, it has long been
known that high concentrations of 2-mercaptoethanol (2-ME) fully
activate IRP1 in vitro (28). Conversely, IRP1 is inhibited
in its ability to bind IREs following treatment with the
sulfhydryl-modifying oxidant diamide or by alkylation (13, 14, 28, 29).
Furthermore, site-directed mutagenesis studies have shown that
Cys437, one of the three cysteines that hold the
iron-sulfur cluster, must be reduced to allow IRP1 to bind IREs (13,
14). As regards IRP2, its regulation mainly proceeds through
proteasome-dependent degradation (16, 30), but recent evidence
indicates that it is also activable to some extent by reducing agents
(26, 31-33). It is thus likely that endogenous redox systems may play
a part in the process of IRP activation. Here, we first investigated the capacity of endogenous reducing systems to cooperate with NO in
IRP1 activation. We focused on two major physiological reducing systems, i.e. reduced glutathione and the thioredoxin
(Trx)/Trx reductase system. Trx is a 12-kDa protein present in many
species from plants to mammals which functions as a major protein
disulfide reductase within cells (34). We found that reduced Trx
strongly enhances the RNA binding activity of IRP1 exposed to NO
donors. In addition, we observed an inhibition of IRP2 activity after exposure to NO in vitro, which was restored by the Trx system.
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EXPERIMENTAL PROCEDURES |
Materials--
3-Morpholinosydnonimine hydrochloride (SIN-1) was
synthesized by Cassella AG (Frankfurt, Germany) and kindly provided by
J. Winicki, Hoechst, France.
S-Nitroso-L-glutathione (GSNO) and diethylamine NONOate (DEANO) were purchased from Cayman Chemical Co. (Ann Arbor, MI). Bovine thioredoxin reductase (TR), Escherichia coli
thioredoxin (Trx), and goat anti-human Trx antibody were from IMCO
Corp. (Stockholm, Sweden). Bovine erythrocyte Cu,Zn-superoxide
dismutase (SOD), insulin, and all other chemicals were from Sigma.
Cell Culture--
The mouse macrophage cell line RAW 264.7 was
obtained from the American Type Culture Collection. Cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc., Paisley, UK) supplemented with 5% low endotoxin fetal calf serum.
Treatment of IRPs--
Mitochondria-free cytosolic extracts were
prepared as described previously (35). In some experiments lysate was
concentrated by ultrafiltration on microconcentrators (Microsep,
Ultrafiltron, Northborough, MA, molecular mass cut-off, 30 kDa).
Cytosolic extract (0.5 mg/ml) was incubated with NO-generating
compounds at 37 °C for different times in 100 mM HEPES,
pH 7.4. In the case of SIN-1, experiments were always performed in the
presence of 3000 units/ml SOD (referred to as SIN-1/SOD). Under these
conditions the production of peroxynitrite, the coupling product of the
reaction between NO and O
2 released by SIN-1, was reduced by
95-98%, as testified by the rhodamine assay (36). Production of
nitrite, one of the end products of NO, was followed to test the
efficacy of the NO donor. Samples treated with NO donors were routinely
desalted on P-6 Bio-Spin chromatography columns (Bio-Rad) prior to
exposure to Trx. Reactions with the Trx system, containing various
concentrations of Trx (oxidized form), 1 nM TR, and 0.4 mM NADPH, were performed at 37 °C for 20 min. These
reaction conditions were sufficient to reduce oxidized Trx fully, as
indicated by the insulin disulfide reduction assay.
Disulfide Reduction Assay--
Disulfide reduction by
thioredoxin was evaluated by the insulin reduction assay (37). Briefly,
0.4 mM NADPH, 1 nM TR, and different
concentrations of Trx were incubated with 9 units/ml insulin in 2 mM EDTA, 80 mM HEPES, pH 7.6, for 20 min at
37 °C in a final volume of 120 µl. The reaction was terminated by
addition of 0.5 ml of 0.4 mg/ml 5,5'-dithiobis(2-nitrobenzoic acid), 6 M guanidine hydrochloride in 50 mM Tris-HCl, pH
8.0, and the absorbance at 412 nm was measured.
Treatment of Recombinant IRP1--
The expression vector for
recombinant IRP1 (rIRP1) has been constructed in the laboratory of Dr.
Lukas C. Kühn (Epalinges, Switzerland). rIRP1 was expressed as a
glutathione S-transferase fusion protein in E. coli and purified on a glutathione-Sepharose column as described
(13). Purified rIRP1 was incubated with NO donors in 10 mM
HEPES, pH 7.6, 40 mM KCl, 3 mM
MgCl2, 5% glycerol, and 50 µg/ml bovine serum albumin
for 1 h at 37 °C. After desalting on a P-6 Bio-Spin column,
rIRP was exposed to the Trx system under the same conditions as
described above.
In Vitro RNA Transcription--
A 32P-labeled IRE
probe was generated by in vitro transcription from the
plasmid pSPT-fer that contains the IRE sequence of human ferritin
H-chain (kindly provided by Dr. L. C. Kühn, Switzerland). Plasmid was linearized by BamHI and transcribed by T7 RNA
polymerase in the presence of 50 µCi of [32P]CTP (NEN
Life Science Products).
Electrophoretic Mobility Shift Assay--
The IRP-IRE
interactions were analyzed as described previously (4, 9) by incubating
2 µg of cytoplasmic protein with a molar excess (0.1 ng = 40,000 cpm) of 32P-labeled ferritin IRE probe in 20 µl of 10 mM HEPES, pH 7.6, 40 mM KCl, 3 mM
MgCl2, and 5% glycerol. IRP·IRE complexes were resolved
on 6% non-denaturing polyacrylamide gels. In parallel experiments,
samples were treated with 2-ME at a final concentration of 2% prior to
the addition of the RNA probe. The IRP·IRE complexes were quantified
with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Electrophoretic mobility shift experiments were performed at least
three times, and one representative experiment is shown.
Aconitase Activity Determination--
The aconitase activity was
measured spectrophotometrically by following the disappearance of
cis-aconitate at 240 nm at 37 °C as described (35).
Nitrite Determination--
The formation of nitrite, one of the
end products of nitric oxide, was determined spectrophotometrically at
543 nm, using the Griess reagent containing final concentrations of
0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine
hydrochloride in 45% acetic acid.
Protein Determination--
The protein content of cytoplasmic
extracts was determined spectrophotometrically at 595 nm, using the
Bio-Rad protein assay with bovine serum albumin as standard.
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RESULTS |
Activation of NO-treated IRP1 by Low Molecular Weight Cytosolic
Factor(s)--
We consistently observed that the IRE binding activity
of IRP1 in cytosolic lysates was little enhanced by treatment with NO
donors like SIN-1/SOD when lysates had been previously concentrated on
a membrane with an Mr cut-off of 30,000 (Fig.
1, lane 2). Furthermore, when
filtrate containing low molecular weight fractions (LF) was added back
to the retentate (HF), the capacity to bind IREs after NO treatment was
largely increased (lane 4). LF had little, if any, effect on
HF from control cell extracts (lane 3). We therefore considered the hypothesis that LF contained a constitutive component with an Mr <30,000 able to activate an
NO-primed pool of IRP1.

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Fig. 1.
A low molecular weight factor increases RNA
binding activity of SIN-1/SOD-treated IRP1. Cytosolic extracts
from RAW 264.7 cells were fractionated through a semi-permeable
membrane into high (HF, >30,000) and low (LF,
<30,000) molecular weight fractions. HF alone or in the presence of LF
was then incubated with 1 mM SIN-1 together with 3000 units/ml SOD for 1 h at 37 °C. Two micrograms of protein were
then analyzed for IRP1·IRE complex formation by an electrophoretic
mobility shift assay (EMSA) with an excess of 32P-labeled
IRE probe in the presence or absence of 2% 2-ME.
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Sensitivity of IRP1 to GSH or Trx after Exposure to NO
Donors--
RAW 264.7 cell cytosol was first incubated with SIN-1
together with SOD. Cytosolic extracts were subsequently exposed to
increasing concentrations of GSH or Trx in the presence of TR and NADPH
and analyzed for IRP1 IRE binding activity. As expected from previous experiments (27), the incubation with SIN-1/SOD for 1 h induced an
approximately 2-fold increase in the IRE binding activity of IRP1
relative to the control (Fig. 2,
A and B, compare lanes 5 and
1). Addition of up to 1 mM reduced glutathione
to cell lysate had only a marginal effect on IRP1 IRE binding. When
lysate had been previously exposed to an NO-generating system, GSH had
a slight but consistent effect at high concentration (
1
mM) (Fig. 2A). In contrast, a strong enhancement
of IRP1 activity (up to 6 times the control value) was observed when
SIN-1/SOD-pretreated cytosolic extracts were exposed to increasing
concentrations of Trx for 20 min (Fig. 2B, see lane
8). Under these conditions, control IRP1 kept its IRE binding
capacity low (lanes 1-4) and aconitase activity high (not
shown). The modulation of IRP1 activity by Trx after exposure to
SIN-1/SOD required electron transfer from NADPH through TR, since
omission of one component of the thioredoxin system, either NADPH, or
TR, or Trx itself, did not induce any further increase in IRP1/IRE
binding (not shown). When Trx was added before or together with the NO
donor, its contribution was not significant. The level of nitrite
released was diminished, suggesting that added Trx partially scavenged
NO.

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Fig. 2.
Sensitivity of IRP1 to GSH and Trx after
exposure to SIN-1/SOD. Cytosolic extracts from RAW 264.7 cells
were concentrated on a membrane with an Mr
cut-off of 30,000 and incubated with 1 mM SIN-1 for 1 h at 37 °C in the presence of 3000 units/ml SOD. Samples were then
desalted on P-6 Bio-Spin columns and treated with increasing
concentrations of GSH or with 1 nM TR, 0.4 mM
NADPH, and increasing concentrations of Trx for 20 min at 37 °C.
IRP1·IRE complex formation was analyzed by EMSA in the presence or
absence of 2% 2-ME. Radioactivity associated with IRP1·IRE complexes
was quantified with a PhosphorImager.
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In a set of time course experiments, cytosolic extracts were incubated
with SIN-1/SOD for increasing periods before exposure to Trx. As can be
seen in Fig. 3, IRE binding activity of
SIN-1/SOD-treated IRP1, which reached 50% of the full activity
expressed in the presence of 2% 2-ME within 30 min, was almost maximal
(90%) when cell cytosol was subsequently exposed to 5 µM
Trx. This enhancement of IRP1 activity induced by Trx in
SIN-1/SOD-treated cytosolic extracts correlated with NO production
which was assessed spectrophotometrically by measuring nitrite
production (Fig. 3, bottom).

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Fig. 3.
Potentiation of IRP1 activation by Trx after
various exposures to SIN-1/SOD. Cytosolic extracts from RAW 264.7 cells were concentrated on a membrane with an Mr
cut-off of 30,000 and treated with 5 mM SIN-1 and 3000 units/ml SOD at 37 °C. At the indicated time points the reaction was
stopped on ice, and the level of nitrites was measured using the
colorimetric assay based on the Griess reaction. After desalting on P-6
Bio-Spin columns, cytosolic extracts were further incubated for 20 min
at 37 °C with 5 µM Trx in the presence of TR and
NADPH. A, equal amounts of protein from each time point were
analyzed for IRE binding by EMSA. B, radioactivity
associated with IRP1·IRE complexes was quantified by PhosphorImaging,
and relative IRP1/IRE binding activity was expressed as a percentage of
the value obtained after exposure to 2% 2-ME alone which allows
visualization of the total binding activity of IRP1. C,
nitrite level after incubation with SIN-1/SOD. , control; , Trx;
, SIN-1/SOD; , SIN-1/SOD + Trx.
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To investigate further if NO is a prerequisite in rendering IRP1
sensitive to Trx, we tested two other types of NO donors that do not
release oxygen-derived reactive molecules: the fast NO-releasing
compound diethylamine NONOate (DEANO), and
S-nitrosoglutathione (GSNO), a product derived from a
physiologically relevant thiol. As shown in Fig.
4A, the IRE binding activity
of IRP1 was moderately increased after incubation with 1 mM
DEANO (27% of the total binding as compared with 10% for non-treated
samples). Exposure to the full components of the Trx system strongly
enhanced IRP1 activity in DEANO-treated samples (compare lanes
4-6, upper). Likewise, addition of Trx to cytosolic extracts
previously exposed to GSNO induced a marked increase in IRP1/IRE
binding, whereas no effect could be detected with GSNO alone (Fig.
3B). Surprisingly, we consistently noted a faster mobility
of the IRP1·IRE complex present in GSNO-treated extracts (lane
4). After exposure to the Trx system, this shift in the
electrophoretic mobility was eliminated in a concentration-dependent manner for Trx (lanes
4-6).

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Fig. 4.
Effect of Trx on IRE binding activity of IRP1
after pretreatment with DEANO or GSNO. Cytosolic extracts were
concentrated on a membrane with an Mr cut-off of
30,000 and incubated with 1 mM DEANO for 20 min at room
temperature or with 5 mM GSNO for 1 h at 37 °C.
Samples were then desalted on P-6 Bio-Spin columns and exposed to the
Trx system as described under "Experimental Procedures." IRP1·IRE
complexes were resolved by EMSA in the presence or absence of 2%
2-ME.
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Effect of Trx on Recombinant IRP1--
To rule out any
interference from other cytosolic components in the activation of IRP1
by Trx, recombinant IRP1 (rIRP1) was purified using a bacterial
expression system and exposed to the Trx system after preincubation
with GSNO or SIN-1/SOD. As shown in Fig.
5, incubation of rIRP1 with GSNO or
SIN-1/SOD slightly increased rIRP1/IRE binding. However, when GSNO- or
SIN-1/SOD-pretreated rIRP1 was further exposed to Trx, IRE binding
activity was dramatically enhanced in a
concentration-dependent manner. Increase in IRE binding
after treatment with Trx did not occur with control rIRP1. These
experiments demonstrate that electrons provided by the Trx system
directly activate the RNA binding of IRP1 previously exposed to NO
generators.

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Fig. 5.
SIN-1/SOD and GSNO increase the sensitivity
of rIRP1 to Trx. Purified rIRP1 was incubated with 5 mM SIN-1 in the presence of 3000 units/ml SOD or with 5 mM GSNO for 1 h at 37 °C. After desalting on P-6
Bio-Spin columns, rIRP1 was incubated with 1 nM TR, 0.4 mM NADPH and increasing concentrations of Trx for 20 min at
37 °C. Samples were analyzed for IRE binding by EMSA, and
radioactivity associated with IRP1·IRE complexes was quantified by
PhosphorImaging. RNA binding activity is expressed as a percentage
of total RNA binding obtained after exposure to 2% 2-ME.
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Endogenous Trx Participates in NO-mediated IRP1 Activation--
To
gain further insights into the role of endogenous Trx in
NO-dependent IRP1 activation, we treated cytosolic lysates
with anti-Trx antibodies before exposure to SIN-1/SOD. As shown in Fig.
6, incubation of cell cytosols with
anti-Trx antibodies abolished the increase of RNA binding activity of
IRP1 routinely observed after treatment with SIN-1/SOD (Fig. 6, compare
lanes 3 and 4). It is therefore clear from this
result that endogenous Trx allows activation of IRP1 in concert with
NO.

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Fig. 6.
Endogenous Trx participates in IRP1
activation by SIN-1/SOD. Cytosolic extracts from RAW 264.7 cells
were incubated with anti-Trx antibodies (0.3 µg/µl) for 30 min at
room temperature prior to exposure to 1 mM SIN-1 together
with 3000 units/ml SOD for 1 h at 37 °C. IRP1·IRE complexes
were then resolved by EMSA in the presence or absence of 2%
2-ME.
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Thioredoxin Reverses IRP2 Inactivation by NO--
RNA binding
activity of IRP2 can be inactivated by treatment with
sulfhydryl-modifying compounds and oxidants (26, 31-33). Therefore, we
investigated whether IRP2 activity could also be affected by NO and
Trx. As shown in Fig. 7, treatment of RAW
264.7 cell cytosol with DEANO decreased IRE binding activity of IRP2 in
a concentration-dependent manner. Neither incubation of
cell cytosol with 500 µM diethylamine nor exposure to
stable end products of DEANO decomposition inhibited IRE binding
activity of IRP2 (not shown). The lower activity of IRP2 upon DEANO
treatment was not a consequence of protein degradation as it could be
restored after incubation with 0.5% 2-ME, which reveals maximal
IRP2/IRE binding (32). Interestingly, IRP2 activity was also restored after exposure of DEANO-treated cell cytosol to 5 µM Trx
in the presence of TR and NADPH. Similar results were obtained with
other NO donors like GSNO (data not shown). It is worth noting that, contrary to what was observed for IRP1, Trx enhanced RNA binding activity of control IRP2.

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Fig. 7.
IRP2 activity is decreased by DEANO and
restored by Trx. Cytosolic extracts from RAW 264.7 cells were
concentrated on a membrane with an Mr cut-off of
30,000 and treated with increasing concentrations of DEANO for 20 min
at room temperature. Samples were then desalted on P-6 Bio-Spin columns
and exposed to 5 µM Trx, 1 nM TR, and 0.4 mM NADPH for 20 min at 37 °C. 32P-Labeled
IRE probe was added to an aliquot of 3 µg of protein, and the
IRP2·IRE complex was analyzed by EMSA. Where indicated, 0.5% 2-ME
was added before the probe to reveal the full extent of IRP2/IRE
binding.
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DISCUSSION |
Relatively little is known about the cellular mechanism that
drives IRPs to modulate translation and turnover of IRE-containing mRNA. As largely documented before, the mode of
activation/inactivation of IRP1 can be explained, at least in part, by
the removal/insertion of a [4Fe-4S] cluster (38). Yet it is well
established that IRP1 activation requires reduction of cysteine
residues (13, 14, 28). Overall, it emerged from these studies that
cluster removal may not be sufficient to give IRP1 the proper
conformation required to fit the stem-loop-shaped IREs. We previously
demonstrated that in response to NO synthesis, IRP1 loses aconitase
activity but gains IRE binding capacity (20). To explain this change, we proposed that NO or a related nitrosating species directly destabilizes the Fe-S cluster of IRP1. As regards IRP2, in
vitro experiments revealed that it is also activable to some
extent by reducing agents (26, 31-33) and inhibited by peroxynitrite, an NO-derived oxidant (32). In this study, we tested the capacity of
two major endogenous reducing systems, i.e. GSH and Trx, to promote or at least participate in concert with NO in IRP activation. First, we found that addition of GSH to macrophage lysate had only a
marginal effect on IRP1/IRE binding in vitro, thus
confirming previous data (29). Moreover, when cell cytosols were
pretreated with NO donors, no significant effect of GSH on IRP1
activity was detected below the millimolar range. These results imply
that modulation of the glutathione pool is not a major parameter which interferes with IRP1 activity. Rather, our data point to reduced thioredoxin as a potent modulator of IRP activity. Trx is a
multifunctional enzyme involved in a vast array of biological functions
ranging from hydrogen donor for ribonucleotide reductase activity to
immunostimulatory effects (39). Yet its main function is a disulfide
reductase activity. By so doing, it allows refolding of disulfide
bridge-containing proteins (40) and modulation of the DNA binding
activity of several redox-sensitive transcription factors including
NF-kB, AP-1 via reduction of Ref-1, glucocorticoid receptor, or heat shock factor-1 (41-45).
Trx by itself had little direct effect on IRP1. The low amount of IRP1
activated directly by reduced Trx in some experiments probably
corresponded to a fraction of apo-IRP1 which spontaneously underwent
oxidation. Interestingly, addition of low concentrations of Trx to IRP1
markedly enhanced the effect of different classes of NO donors on IRE
binding. It is noteworthy that GSNO had two effects on IRP1 as follows:
(i) like the other NO donors, it predisposed IRP1 to IRE binding upon
further reduction, and (ii) it yielded a modification in the protein
characterized by a downward shift in the polyacrylamide gel migration.
Migration returned to normal after exposure to 2% 2-ME or 10 µM Trx. It is tempting to speculate that
trans-nitrosation mediated by GSNO promoted formation of two different
disulfide bridges. One would mostly affect binding to IRE by oxidizing
at least one allosteric thiol close or belonging to the IRE-binding
domain. Another disulfide bond, selectively formed by GSNO, may affect
vicinal thiols distal from the IRE-binding domain. The structural
change that may result from the latter does not seem to be crucial for
activity as it did not affect RNA binding capacity and may not be
effective in the presence of reduced Trx. Besides, we observed that
sequential exposure of IRP1 to NO and reduced Trx led to the largest
increase in IRP1/IRE binding. This is probably due to the fact that Trx
must be maintained reduced by TR and NADPH. Such a situation was
previously encountered with H2O2-enhanced DNA
binding of heat shock factor-1 (45). It is likely that combining an NO
generator with exogenously added Trx can partially overcome the
reducing capacity of the latter. Indeed, as pointed out by Nikitovic
and Holmgren (46), reaction of GSNO with Trx leads to homolytic
cleavage with release of NO and inhibition of the TR/Trx system.
Further evidence for a physiological role of Trx in IRP1 activation was
provided by the use of specific anti-Trx antibodies. Indeed,
neutralization of endogenous Trx prevented NO-mediated activation of
IRE binding by IRP1.
Enhancement of IRP1 RNA binding activity in response to high
concentrations of reducers such as dithiothreitol or 2-ME has long been
known (28), but intriguingly neither low levels of these chemicals nor
physiologically relevant reducers have been shown to reproduce this
effect. The major obstacle to mild reduction is probably the presence
of the Fe-S cluster which, in partnership with substrate
(e.g. citrate), prevents access to the sensitive thiols,
i.e. Cys437, Cys503 and
Cys506 (13). Even at high concentrations, 2-ME is not able
to remove the cluster since after filtration on a G-25 column, IRP1
still needs a high concentration of 2-ME to bind IREs (13), which is
characteristic of a cluster-containing form of IRP1. In fact, IRP1
becomes readily sensitive to low amounts of 2-ME only after cluster
removal (13). Apart from the two classical forms of IRP1,
i.e. the [4Fe-4S]-IRP1 (aconitase) and the apo-IRP1
(IRE-binding regulator), a [3Fe-4S] cluster-containing IRP1 was
described (47, 48). In addition, recent evidence from several
laboratories including ours has indicated that an oxidized form of
apo-IRP1 exists in vitro (32, 49, 50) and in living cells
(26). This form of IRP1 has been assigned to a cluster-free protein whose oxidation of sulfhydryl groups at the active site confers on it
neither aconitase activity nor IRE binding capacity. We recently showed
that such an IRP1 also exists after exposure to peroxynitrite (32). In
this form of IRP1, oxidation of Cys437 by peroxynitrite
would prevent IRE binding. We proposed that this oxidized apo-IRP1 is a
latent form rapidly available to bind IREs upon physiologic reduction
(32). However, thus far the nature of this reduction has remained
enigmatic. The data presented here suggest that reduced Trx acts in
partnership with NO or NO-derived oxidizing species to activate IRP1
within cells. Moreover, the fact that Trx activates a pool of IRP1
previously exposed to NO provides an explanation for a somewhat
paradoxical situation. Indeed, NO is a signaling molecule that, in the
presence of oxygen, can yield oxidizing or nitrosating reactive species
which form S-nitrosothiol adducts and/or disulfides on
proteins. Higher oxides derived from NO, like peroxynitrite, may also
yield sulfenic or sulfinic groups (51). It was therefore intriguing
that NO or related products alone could activate IRP1, which needs to
be reduced prior to binding IREs. Whether critical sulfhydryls of IRP1
are S-nitrosylated or oxidized to sulfenic or sulfinic acids in NO-producing cells remains to be determined, but since the disulfide-reducing Trx was able to generate full RNA binding activity, the most likely conclusion is that nitrogen oxides, in addition to
disrupting the cluster, also interact with the cluster-coordinating cysteines Cys503, Cys506, and
Cys437 to promote a disulfide bridge. We anticipate that
such a bridge should link Cys437 and either
Cys503 or Cys506. Our results point to the Trx
system as the most effective reducing system ultimately to activate
such an oxidized apo-IRP1.
IRP2 has the same specificity as IRP1 and, despite the fact that it is
generally less abundant than IRP1 (52), it can regulate cellular iron
metabolism by itself, as recently shown in IRP1-lacking cells (53).
Moreover, its effect on ferritin expression may prevail (54), and it
has been recently proposed that IRP2 rather than IRP1 is involved in
abnormal iron distribution in the brains of patients suffering from
Alzheimer's disease (55). IRP2, in contrast to IRP1, does not possess
an Fe-S cluster but has the 3 corresponding cysteines which in IRP1
anchor the cluster and, remarkably, 5 cysteine residues in its specific
insertion sequence (16). Its activity was enhanced in vitro
after exposure to 2-ME or dithiothreitol (26, 31-33) and in
vivo, in a model of acute inflammation (56). As macrophages can
produce both NO and large amounts of reactive oxygen species, it is
therefore relevant to consider carefully the effect of physiological
reactive mediators such as NO on IRP2. Here, we showed an inhibition of
RNA binding of IRP2 in macrophage cytosol following exposure to NO
donors. Inhibition was reversed by reduced Trx but not by GSH.
Trx/TR-dependent reduction of intracellular disulfide may
therefore regenerate IRP2/IRE binding activity of macrophages exposed
to situations where oxidizing species can be generated, e.g.
when they migrate into inflammatory areas or secrete oxidants during
phagocytosis or after stimulation by chemotactic factors.
In conclusion, these findings provide a decisive clue to the regulation
of IRPs. We propose that NO and Trx cooperate to regulate IRP
function(s). A few examples already exist where the NO/Trx tandem has
been shown to modulate efficiently enzyme or transcription factor
activity (57, 58). It is likely that Trx-catalyzed disulfide reduction
of IRPs previously oxidized by nitrogen or oxygen-derived species
represents a novel example of coordinated regulation of gene expression.