©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of Iron Regulatory Protein Functions
FURTHER INSIGHTS INTO THE ROLE OF NITROGEN- AND OXYGEN-DERIVED REACTIVE SPECIES (*)

(Received for publication, August 31, 1995; and in revised form, November 15, 1995)

Cécile Bouton Martine Raveau Jean-Claude Drapier (§)

From the From U 365 INSERM, Section de Recherche, Institut Curie, 26, rue d'Ulm, 75231 Paris Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Iron regulatory protein (IRP) is a cytosolic bifunctional [Fe-S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA. The modulators of these activities are the intracellular concentration of iron and, as recently described, NO synthase activity. In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO, the product of the reaction between NO and O(2)), as well as oxygen-derived radicals (O(2) and H(2)O(2)) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophage-like RAW 264.7 cells. Neither the addition of a bolus of ONOO or H(2)O(2) nor O(2) generation significantly affected IRE binding even though they inhibited its aconitase activity. Moreover, we show that 3-morpholinosydnonimine (SIN-1), a chemical which releases both NO and O(2), enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus glutathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, an aconitase substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O(2) nor ONOO can convert [4Fe-4S] IRP into IRE-binding protein but rather suggest that an active redox form of NO converts IRP into its IRE binding form by targeting the [Fe-S] cluster.


INTRODUCTION

Iron regulatory protein (IRP) (^1)controls iron homeostasis by regulating the expression of ferritin and transferrin receptor through binding to iron-responsive elements (IREs) in the 5`- and 3`-untranslated regions of their respective mRNAs. In iron-depleted cells, IRP has high affinity for IRE(s) and both stabilizes transferrin receptor mRNA and prevents ferritin translation. Conversely, in iron-replenished cells, it functions as an [Fe-S] cluster-containing aconitase which converts citrate into isocitrate in the cytosol(1, 2) . These activities are mutually exclusive, because the fully assembled [4Fe-4S] cluster required for enzymatic activity at the active site sterically inhibits IRE binding (2, 3) . We previously reported that induction of nitric-oxide synthase in activated murine macrophages results in loss of mitochondrial enzyme activity(4, 5) . In addition, we showed that inhibition of mitochondrial aconitase was rapidly reversible(6) . Subsequently, we and others demonstrated that NO synthesis increases IRE binding in several cell types(7, 8, 9) . Conversely, NO synthesis causes loss of aconitase activity of IRP as shown for mitochondrial aconitase(7, 9) . Therefore, the functional behavior of IRP, either aconitase or IRE-binding protein, can be determined by the intracellular iron concentration (1, 2) and the NO flux into the cell(7, 8, 9, 10) . Another line of research showed previously that the activities of bacterial and mammalian aconitases are inhibited by O(2)(11, 12, 13) , whereas the activity of plant aconitase which exhibits a strong homology with mammalian IRP (14) is inhibited by H(2)O(2)(15) . As loss of the aconitase activity of IRP generally correlates with an increase in IRE binding(2, 16) , it seems reasonable to postulate that reactive oxygen intermediates (ROS) enhance IRE binding of IRP as well. Further, more recent findings support the view that peroxynitrite (ONOO), a reactive molecule derived from NO, is responsible for inhibiting both cytosolic and mitochondrial aconitases, rather than NO itself(12, 17) . All these results and considerations led us to investigate IRE binding by IRP to ascertain whether it is directly sensitive to O(2), H(2)O(2) (ROS), and ONOO. For this purpose, we compared the direct effect of these three species on IRP activities to that of various NO donors on IRP activities. The results showed that even though ROS and ONOO inhibited aconitase activity of IRP, contrary to NO donors, they did not activate its IRE binding activity.


EXPERIMENTAL PROCEDURES

Materials

S-Nitroso-L-glutathione (GSNO) was purchased from Alexis (Switzerland). S-Nitrosothiol derived from cysteamine and referred to as S-nitrosocysteamine (SNC) was kindly provided by Pr. Marc Fontecave, Grenoble, France. Spermine-NONOate (Sper/NO) was from Cayman Chemical Co. (Ann Arbor, MI). Dihydrorhodamine was purchased from Molecular Probes, Leiden, Holland. 3-Morpholinosydnonimine hydrochloride (SIN-1) and its end product SIN-1C were synthesized by Cassella AG (Frankfurt, Germany) and kindly provided by J. Winicki, Laboratoires Hoechst, France. Hydrogen peroxide was from Merck (Darmstadt, Germany). Xanthine oxidase (720 milliunits/mg), catalase, bovine erythrocyte Cu,Zn-superoxide dismutase (SOD), bovine hemoglobin, and all other chemicals were from Sigma. Peroxynitrite (ONOO) was synthesized as described previously (18) and concentrated by freezing. The concentration was determined spectrophotometrically at 302 nm ( = 1670 M cm).

Treatment of IRP

Mitochondria-free cytosolic extracts were prepared from the RAW 264.7 macrophage-like cell line as described previously(6) . Briefly, cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) to which 5% low endotoxin fetal calf serum had been added, harvested, washed, suspended in 0.25 M sucrose buffered with 100 mM HEPES, pH 7.4 (50 times 10^6/ml), and treated with 0.007% digitonin for 5 min at 4 °C. The resulting lysate was then centrifuged at 75,000 rpm for 20 min in a TL 100 Beckman ultracentrifuge. The cytosolic extract (approx1 mg/ml protein) was incubated with NO-generating compounds at 37 °C for 1 h in either 40 µl of 10 mM Hepes, pH 7.6, 40 mM KCl, 3 mM MgCl(2), and 5% glycerol, or in PBS. 2-3 µg of protein and 100 µg were analyzed for IRE binding activity and aconitase activity, respectively. Nitrite, one of the end products of NO, was measured to test the efficacy of the NO donor. Cytosolic extracts were also exposed to O(2) generated by 150 µM hypoxanthine and 5.5 milliunits/ml xanthine oxidase in the presence or absence of 300 units/ml SOD or to freshly prepared dilutions of H(2)O(2) in 40 µl. Peroxynitrite (up to 1 mM, final concentration) was diluted in 0.1 M NaOH, and 2 µl was applied to the inside of the cap of an Eppendorf tube. The tubes were closed and the solutions were mixed by quickly spinning the tube containing IRP in 200 mM Tris-HCl buffer, pH 7.4, 40 µl final volume. In control tubes, peroxynitrite was added to buffer 5 min before protein (reverse order addition control) in order to let it decompose at neutral pH before reacting with IRP. In some experiments, samples were desalted on P-6 Bio-Spin chromatography columns (Bio-Rad) prior to analysis.

Gel Mobility Shift Assay

Gel electrophoresis of IREbulletprotein complexes was performed as described previously(19, 20) . Briefly, a P-labeled IRE probe was transcribed in vitro from the plasmid pSPT-fer which contains the sequence corresponding to the IRE of ferritin human H-chain (kindly provided by Dr. L. C Kühn, Switzerland). Two µg of the linearized plasmid was transcribed in vitro by T7 RNA polymerase. The DNA template was removed by digestion with RNase-free DNase I. The IRE probe was then extracted by phenol-chloroform (1:1, v/v). Three µg of cytoplasmic protein was mixed with 0.1 ng of [P]CTP-labeled IRE protein in 40 µl of 10 mM Hepes, pH 7.6, 40 mM KCl, 3 mM MgCl(2), and 5% glycerol (20 µl total volume). After a 20-min incubation at room temperature, RNase T1 (1 unit/µl) was added and, 10 min later, 2 µl of 50 mg/ml heparin. After 15 min, proteins were run on nondenaturing 6% acrylamide gel. In a parallel experiment, samples were routinely treated with 2-mercaptoethanol (2-ME) at a final concentration of 2%, prior to addition of the IRE probe to allow full expression of IRE binding activity(21) . Gels were dried and exposed to Amersham Hyperfilm MP film at -80 °C. The IRPbulletIRE complex was quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Band shift experiments were performed at least three times, and one representative experiment is shown.

Oxyhemoglobin Preparation

Hemoglobin (3 mM) in PBS was treated with dithionite and oxygenated by vigorous shaking. Excess dithionite was removed by two filtrations on columns prepacked with G-25 (Pharmacia). The oxyhemoglobin spectrum was then recorded.

Aconitase Determination

The aconitase activity of cytosolic extracts was determined spectrophotometrically, by measuring the disappearance of cis-aconitate at 240 nm as described previously(6) . Units represent nanomoles of substrate consumed/min at 37 °C ( = 3.6 mM cm).

Nitrite and Nitrate Determination

The formation of nitrite, one of the oxidation 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. Nitrate was reduced to nitrite by nitrate reductase from Aspergillus niger (Sigma), prior to measurement by the Griess reagent.

Superoxide Determination

The release of superoxide from the generating system hypoxanthine (2 times 10M)/xanthine oxidase (10 units/ml) was measured spectrophotometrically by monitoring the SOD-inhibitable reduction of 160 µM ferricytochrome c at 550 nm at 37 °C ( = 21 mM cm). One mol of O(2) reduces 1 mol of ferricytochrome c.

Peroxynitrite Determination

Authentic ONOO or ONOO released from SIN-1 was assessed spectrophotometrically by monitoring the oxidation of dihydrorhodamine at 500 nm as described previously(22) .

Protein Determination

The protein content of cytoplasmic extracts was determined spectrophotometrically at 595 nm, using the Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as standard.


RESULTS

Aconitases are [Fe-S] enzymes sensitive to oxidation. Even though aconitase activity of IRP is more stable than that of mitochondrial aconitase, it tends to decrease progressively with time when exposed to air. Likewise, IRE binding activity also responds to the effects of oxidoreduction. Hence, in accordance with the results of preliminary experiments, we avoided incubating IRP too long in vitro. Cell lysates were therefore exposed to additives for 1 h or less before the activities of IRP were determined. IRE binding activity was analyzed by gel shift experiments, and, in some experiments, the aconitase activity of IRP was also measured by spectrophotometry. In our experiments, two bands corresponded to specific IRE-binding proteins since they were displaced by an excess of specific IRE probe (not shown): a main band, indicated by an arrow, and a faint and somewhat elusive faster migrating band. This second IRP (called IRP(B) or IRP2, to distinguish it from the main band IRP, designed IRP1) was recently characterized(23, 24, 25, 26) . This second IRP has no aconitase activity, and it is still not clear whether it possesses an [Fe-S] cluster(24) , but, according to a recent report, it also responds to iron starvation and to NO synthesis(26) . However, IRP2 is far less stable than IRP1 and, contrary to the latter, its ability to bind IRE does not result from a post-translational mechanism(27) . In our system, IRP2 exhibited little activity and did not respond consistently to any stimulus applied. Therefore, in the results given below, only IRP1, referred to as IRP, will be considered.

SIN-1 is an NO generator commonly used in vivo as a vasodilator. It also releases O(2) which reacts with NO to yield ONOO(28, 29, 30) . Therefore, SOD, an O(2) scavenger, is usually added along with SIN-1 to increase the NO half-life. As shown in Fig. 1A, the combination of SIN-1 plus SOD significantly increased IRE binding by IRP with time. According to phosphorimaging quantification, IRE binding activity by IRP from SIN-1/SOD-treated cytosol reached up to 50% of the full activity expressed in the presence of 2% 2-ME within 1 h. Concomitantly, aconitase activity dropped drastically (Fig. 1B). In another set of experiments, cytosolic extracts were also exposed to increasing concentrations of SIN-1 (plus SOD) for 30 min. Nitrite and nitrate, taken as indicators of NO production, were released by 0.5, 1, 5, and 10 mM SIN-1 at constant rates of 1, 1.6, 4.3, and 6 µM/min, respectively. Fig. 2shows that, during this incubation, IRE binding activity markedly increased with the amounts of SIN-1 applied. Neither SIN-1C, which is a by-product of SIN-1 unable to release NO, nor nitrite and nitrate had any effect (not shown).


Figure 1: Time course of the modulation by SIN-1 of aconitase and IRE binding activities of IRP in RAW 264.7 cell cytosol. Cytosol (20 µg of protein) was treated with 1 mM SIN-1 and 750 units/ml SOD for increasing periods of time at 37 °C. The reaction was stopped on ice. A, P-labeled human ferritin H-chain IRE probe (60,000 cpm) was added to an aliquot of 2 µg of protein, and the IREbulletIRP complex was analyzed by gel retardation as described under ``Experimental Procedures.'' Where indicated (bottom lanes), 2% 2-ME was added before the probe to reveal the full extent of IRE binding. Sections of autoradiograms of native gel are shown, and the position of the free probe was cut off from the bottom of the autoradiogram. The IREbulletIRP complex is visualized by an arrow. B, IRE binding activity quantified by phosphorimaging and expressed as percent of the value obtained with 2% 2-ME. % IRE binding activity, as well as aconitase activity, were plotted versus the time of cell cytosol exposure to SIN-1/SOD. Measurement of aconitase activity (100 µg of protein) was based on the disappearance of cis-aconitate, detected at 240 nm. One unit is defined as nanomoles of cis-aconitate consumed per min.




Figure 2: Dose dependence of the enhancement of the IRE binding activity of IRP upon SIN-1 treatment of RAW 264.7 cell cytosol. This cytosol (50 µg of protein) was treated with increasing doses of SIN-1 for 30 min in the presence of 750 units/ml SOD, and IRE binding was analyzed as in Fig. 1.



The rate constant of the reaction between NO and O(2) was re-evaluated recently (30) and was found to be similar to that of the dismutation of O(2) by SOD: 6.7 times 10^9M sversus 2 times 10^9M s(31) . Accordingly, it is likely that even in the presence of modest amounts of SOD, SIN-1 releases O(2) which, by combining with NO, yields ONOO(29, 30) . This is the reason why we decided to compare carefully the IRE binding of IRP exposed to SIN-1 either alone or along with large amounts of SOD (3000 units/ml), in order to make certain that NO was produced in larger amounts than O(2)(30) . As testified by the rhodamine assay (22) , ONOO release from SIN-1 was reduced by 95-98% in the presence of 3000 units/ml SOD. As shown in Table 1, 1 mM SIN-1 inhibited the aconitase activity of IRP either in the presence of SOD or in its absence, but catalase abrogated the inhibitory effect of SIN-1/SOD. These results are consistent with the fact that O(2) and ONOO, two products released from SIN-1, as well as H(2)O(2), are known to inhibit aconitase activity(12, 15, 17) . Regarding IRE binding, SIN-1 by itself was hardly active at all but note that it was far more active in the presence of 3000 units/ml SOD (Fig. 3). The potency of the combination SIN-1/SOD was not due to increased production of NO as judged by nitrite and nitrate release (not shown). Further, the addition of catalase reduced IRE binding only slightly, thus suggesting that H(2)O(2) which might be produced by the combination SIN-1/SOD, did not play a major part in this play. Oxyhemoglobin, which binds NO with high affinity, protected IRP against the effect of SIN-1/SOD in a dose-dependent manner (Fig. 4), thus confirming that any O(2) released from SIN-1 cannot per se make IRP bind IRE.




Figure 3: The effect of SOD on the IRE binding activity of IRP exposed to the NO donor SIN-1. RAW 264.7 cell cytosol was treated with 1 mM SIN-1 for 1 h at 37 °C in the presence of 3000 units/ml SOD and 100 units/ml catalase in PBS or in their absence. IRE binding was then analyzed as in Fig. 1.




Figure 4: The effect of hemoglobin on the IRE binding activity of IRP induced by the NO donor SIN-1. RAW 264.7 cell cytosol was treated with 5 mM SIN-1 for 1 h in the presence of 3000 units/ml SOD (lanes 3-7) and increasing amounts of hemoglobin (lanes 4-7). Samples were then desalted on P-6 Bio-Spin columns and analyzed for IRE binding activity as in Fig. 1.



Aconitase substrates such as citrate and cis-aconitate bind to an [4Fe-4S] cluster at the active site of aconitases (16, 32) and protect IRP from the effect of 2-ME(33) . To see whether the reactive molecule released by SIN-1 targets the iron-sulfur center of IRP, we incubated cell cytosol with both SIN-1 (plus SOD) and cis-aconitate and then measured both aconitase and IRE binding. The results (Fig. 5A) show that the presence of cis-aconitate prevented SIN-1 from inducing IRP to bind IRE. As previously documented(33) , cis-aconitate also protected IRP from the effect of 2-ME (Fig. 5A, bottom lanes). In another set of experiments, incubation of cis-aconitate with SIN-1 also prevented the inhibition of aconitase activity (Fig. 5B).


Figure 5: The effect of cis-aconitate on the activities of IRP exposed to SIN-1. RAW 264.7 cell cytosol was incubated for 30 min at 37 °C with 5 mM SIN-1 and 750 units/ml SOD along with cis-aconitate where indicated. Samples were then analyzed for IRE binding activity (A) or desalted on P-6 Bio-Spin columns and analyzed for aconitase activity (B), as described in Fig. 1.



ROS inhibit aconitase activity in various systems (13, 15) and interact with the [Fe-S] cluster of several dehydratases(34) . These species were therefore good candidates for converting IRP from the aconitase form to the IRE binding form. To test this possibility, RAW 264.7 cell cytosol was exposed either to the O(2) generator system hypoxanthine/xanthine oxidase or directly to H(2)O(2). Under these conditions, the hypoxanthine/xanthine oxidase system generated significant flow of O(2) (3 to 5 µM/min for at least 15 min) as determined by the SOD-inhibitable reduction of ferricytochrome c. H(2)O(2) was probably also produced following spontaneous dismutation of O(2). IRP lost its aconitase activity upon the generation of ROS (Table 2), and inhibition of this activity was completely prevented by the addition of both SOD and catalase. Regarding IRE binding, Fig. 6shows that exposure of IRP to the O(2) generator system had no significant effect (left panel). Likewise, direct exposure of cell cytosol to high doses of H(2)O(2) reduced aconitase activity by about 50% (Table 2) without increasing the IRE binding activity of IRP (Fig. 6, right panel). In contrast, in both sets of experiments, the combination of SIN-1 plus SOD, taken as a positive control, significantly increased IRE binding by IRP (Fig. 6, lanes 6 and 12, respectively).




Figure 6: Comparative effects of ROS and of the NO donor SIN-1 on the IRE binding activity of IRP. RAW 264.7 cell cytosol was incubated for 1 h at 37 °C with the hypoxanthine/xanthine oxidase O(2) generating system (left panel) or with H(2)O(2) (right panel). In the same sets of experiments, cytosol was also exposed to 2 mM SIN-1 (left panel) or 5 mM SIN-1 (right panel) to which 3000 units/ml SOD had been added. Samples were then analyzed for IRE binding activity as described in Fig. 1.



When cell cytosol was exposed to a directly applied bolus of ONOO, the aconitase activity of IRP was significantly inhibited by ONOO concentrations from around 25 µM (Fig. 7A), thus confirming previous published data(12, 17) . However, Fig. 7B shows that at concentrations of up to 250 µM, ONOO had no effect on IRE binding. In several other similar experiments, ONOO, even at 1 mM, had no effect either. Yet in parallel experiments, ONOO was able to oxidize dihydrorhodamine into rhodamine (not shown). These experiments indicate that ONOO was able to inhibit IRP aconitase activity significantly but did not affect its IRE binding activity.


Figure 7: Effect of ONOO on IRP activities. RAW 264.7 cell cytosol was incubated for 10 min at room temperature with increasing concentrations of ONOO. In the first set of experiments (A), aconitase activity was assessed spectrophotometrically by measuring the initial rate of disappearance of cis-aconitate at 240 nm. Experiments were representative of 4 which gave similar results. In the second set of experiments (B), cytosol incubated with ONOO was analyzed for IRE binding using gel retardation as in Fig. 1. Where indicated, peroxynitrite was allowed to decompose in buffer before the cytosol was added (reverse-order addition control or ROA).



Besides SIN-1, two other types of chemicals that release NO but not oxygen-derived molecules were tested for their capacity to enhance IRE binding by IRP: S-nitrosothiols and the secondary aminebulletNO complex, spermine-NONOate (sper/NO). Two S-nitrosothiols were routinely assayed: S-nitrosoglutathione (GSNO), a product derived from physiologically relevant thiol, and an S-nitrosothiol derived from cysteamine and further referred to below as S-nitrosocysteamine (SNC). GSNO is fairly stable and required the presence of a reducing agent, e.g. ascorbate, to release significant amounts of NO (as testified by nitrite production) during its 1-h incubation with IRP. SNC rapidly decomposes into NO and is kinetically predictable. Indeed, Roy et al.(35) reported that millimolar range of SNC rapidly yields micromolar concentrations of NO. SNC was therefore suitable for short-time incubation with IRP. Under our conditions, GSNO (+ ascorbate) and SNC released approx4 and 8 µM nitrite/min, respectively, during the 1-h incubation. Fig. 8shows that both S-nitrosothiols made IRP bind IRE. S-Nitroso-DL-penicillamine, another well-known S-nitrosothiol, was also assayed but gave, with time, gave inconsistent results, and the experiments were not pursued. We also used sper/NO, another fast NO-releasing compound(36) . Half-time of sper/NO is approx 40 min at pH 7.5, so that it was suitable for a 1-h incubation with IRP. Exposure of cytosol to 1 mM sper/NO consistently inhibited aconitase activity by 88% (mean of 6 experiments). Inhibition was prevented by oxyhemoglobin (not shown). The results depicted in Fig. 9(lane 6) show that sper/NO increased IRE binding by IRP moderately, but did so far more in the presence of reduced glutathione (GSH) (lane 7). Ascorbate also boosted the capacity of sper/NO to activate IRE binding activity of IRP (not shown). It was interesting to observe that oxyhemoglobin prevented the increase in IRE binding induced by the combination of sper/NO and GSH (lane 8), but methemoglobin did not (lane 9).


Figure 8: Effect of nitrosothiols on IRE binding activity of IRP. RAW 264.7 cytosol (30 µg of protein) was incubated for 1 h at 37 °C with SNC (left panel) or 5 mM GSNO plus 1 mM ascorbate (right panel). Samples (2 µg) were then analyzed for IRE binding, with or without 2% 2-ME, as described in Fig. 1.




Figure 9: The effect of sper/NO on the IRE binding activity of IRP. RAW 264.7 cell cytosol was incubated for 1 h at 37 °C with 1 mM sper/NO, either alone (lane 6) or together with 1 mM glutathione (lane 7), and either 100 µM oxyhemoglobin (lane 8) or 100 µM methemoglobin (lane 9). The cytosol was also treated with 1 mM spermine (Sper, lane 5) or 1 mM spent sper/NO, i.e. sper/NO which had been allowed to decompose for several hours before its addition to the cytosol (lane 10).




DISCUSSION

Several lines of evidence indicate that as well as oxygen-derived molecules, NO, which diffuses freely away from its site of generation, may be a biological messenger used by the cell to react with redox-sensitive gene trans-regulators. Thus, it was reported that exposure to O(2) or H(2)O(2) or to exogenous NO activates of the eukaryotic trans-activators AP-1 (37, 38) and NF-kappaB(39, 40) , as well as the bacterial Sox-RS regulon system(41, 42) . Furthermore, we and others previously supplied evidence that endogenous and exogenous NO modulates the cellular iron metabolism by increasing IRP binding to specific sequence(s) in their mRNA, i.e. to IREs(7, 8, 10, 26, 43) . Conversely, the aconitase activity of IRP is lost as a consequence of NO synthesis. The redox-sensitive site of IRP responsive to NO synthase product(s) is probably a [4Fe-4S] cluster which when intact, both allows enzymatic activity and prevents IRE binding(2, 3, 16) . Besides, O(2) and ONOO were identified as likely products of brain NO synthase(43, 44) . In addition, O(2) is known to interact with iron-sulfur clusters and has been reported to inhibit aconitase in E. coli and mammals(11, 13, 33) whereas H(2)O(2) inhibits plant aconitase(15) . Recently, it was further proposed that O(2) represses ferritin translation by activating IRE binding through the interaction of IRP with IRE(46) . Consequently, it was plausible to envisage that either of these two products could be responsible for the converse modulation of IRP exhibited by cells expressing NO synthase. Accordingly, to gain insight into the physiologically relevant mechanism that allows NO-producing cells to express IRP, crude IRP from macrophage extracts was exposed to ROS, ONOO, and various NO-releasing compounds, to see whether these species increased IRP binding to IRE and, if so, which one(s).

In the presence of oxygen, SIN-1 was found to release both NO and O(2)(28) and, in the absence of SOD, it is often considered as a source of ONOO(29, 47) . It is therefore a useful tool for studying the effect of ONOO produced at a low rate and by adding SOD and catalase, the respective contributions of O(2) and NO to the effect of SIN-1. We showed here that incubation of IRP with SIN-1 decreased its aconitase activity. Inhibition was completely abolished by a combination of SOD and catalase, suggesting at first glance that H(2)O(2) had a role in this inhibition. However, direct addition of high doses of H(2)O(2) to cell cytosol did not completely inhibit aconitase activity. Taken together, these results imply that H(2)O(2) alone is not sufficient to inhibit the aconitase activity of IRP and may require a combination with NO to be potent. Such interplay between H(2)O(2) and NO has already been suggested to play a part in cell cytotoxicity(48, 49) . SIN-1 alone did not increase IRE binding by IRP significantly, but it must be emphasized that the presence of SOD consistently increased the IRE binding of IRP, thus confirming our previous results obtained with recombinant human IRP (7) . The reason why SOD enhances the SIN-1-stimulated IRE binding activity is still not clear. The simplest explanation is that SOD prolongs NO half-life by preventing its reaction with O(2). Alternatively, SOD has been suggested to favor the conversion of NO into certain redox forms independently of dismutation(50, 51) . Here, the presence of catalase in addition to SOD led to a slight but consistent reduction of IRE binding. However it is doubtful that H(2)O(2) was directly involved in this reduction because addition of large doses of H(2)O(2) had no significant effect on IRE binding activity of IRP. Further, these results are consistent with those of Pantopoulos and Hentze (52) who recently showed that even though H(2)O(2) enhanced IRE binding in a fibroblast cell line, it had no direct effect on this activity. It is possible that the protection afforded by catalase of aconitase activity and IRE binding against SIN-1 plus SOD was due to the scavenging of nitric oxide by catalase, a heme-containing enzyme.

Aconitase substrates stabilize the [Fe-S] cluster of mitochondrial aconitase and presumably that of IRP (3, 16, 32) by binding to one iron atom, known as Fe(a). We showed here that cis-aconitate attenuated the increase in IRE binding induced by SIN-1 plus SOD. The protective effect of cis-aconitate is thus consistent with the proposal that a SIN-1-derived reactive molecule and cis-aconitate compete for the same site of ligation, i.e. the [Fe-S] cluster. Further, the protective effect of citrate raises the possibility that the cellular concentration of citrate may exert a feedback mechanism toward the stimulation by NO synthase of IRE binding.

The exposure of cell cytosol to the generation of ROS by the hypoxanthine/xanthine oxidase system led to the inhibition of IRP aconitase activity without allowing IRP binding to IRE. However, when SOD, even at high doses, was added in parallel experiments, SIN-1 greatly enhanced IRE binding. The fact that the O(2) generating system was innocuous and that the presence of SOD increased the effect of SIN-1 rules out the possibility that O(2) in itself makes a major contribution to the switch of IRP into IRE-binding protein.

In addition, there is growing evidence that ONOO, which results from the reaction of O(2) with NO, may play an important part in several pathophysiological effects formerly attributed to NO(53) . In the same line of research, it was shown that ONOO is produced by rat alveolar macrophages triggered in vitro by phorbol 12-myristate 13-acetate (54) and can inhibit the mitochondrial respiration complexes I and II containing [Fe-S] clusters(55) , as well as both mitochondrial and cytosolic aconitase(12, 17) . Consequently, it has been proposed that ONOO, rather than NO, is the physiological effector molecule which in cells expressing NO synthase activity, inhibits aconitase activity. As aconitase activity and the IRE binding activity of IRP are reciprocally modulated in response to changes in the iron concentration (2) or to NO synthesis(7, 10) , it was tempting to conclude that ONOO increases the IRE binding activity of IRP. Our results provided evidence that this is unlikely because ONOO when applied as a bolus or released from SIN-1 has little or no effect on IRE binding by IRP. One may wonder whether absence of effect of ROS and ONOO resulted from absence of an adequate ``redox status'' in the cell extract. This deserves further investigation, but the marked difference between the effect of SIN-1 alone (which releases ONOO) and SIN-1 plus large amounts of SOD (which release NO) seems to call for another explanation.

Lastly, to improve our knowledge of the precise mechanism by which the NO synthesis can change the functional response of IRP, we exposed cell cytosol to two S-nitrosothiols which release NO but not O(2): exposure to SNC and GSNO (the latter in the presence of ascorbate), resulted in enhancement of IRE binding. GSNO was previously shown by Castro et al.(17) to inhibit aconitase activity, but these authors concluded that the release of NO was not responsible for this inhibition, and S-nitrosothiols indeed react with protein thiols, e.g. by trans-nitrosation or by thiyl radical release, independently of their capacity to yield NO. Therefore, another chemical able to release NO was also tested, the ``NONOate'' sper/NO. Provided it was used with GSH, sper/NO gave results similar to those obtained with S-nitrosothiols. The boosting effect of reducers in triggering IRE binding was not evaluated, but it was not related to an enhanced capacity to release NO, as testified by nitrite production. Rather, it suggests that an adequate redox environment within the cell favors IRE binding resulting from NO production. Further, since oxyhemoglobin, an NO-scavenger, reduced IRE binding resulting from the action of sper/NO plus GSH, we inferred that the formation of the IRPbulletIRE complex resulted from NO release rather than to a side effect of sper/NO.

The fact that ROS and ONOO inhibit the enzymatic activity of IRP without triggering IRE binding may raise an important issue. These products may affect the iron-sulfur cluster of IRP only to a limited extent, so that they only convert the [4Fe-4S] form, which allows aconitases to be active, into the [3Fe-4S] form which possesses neither aconitase activity nor IRE binding ability(2, 3, 16) . Alternatively, we cannot exclude the possibility that ONOO both disrupts the cluster and prevents binding to IRE, by promoting the oxidation of some free sulfhydryl groups required for binding IREs(3, 21) . It is noteworthy that two cysteine residues involved in iron-sulfur cluster assembly, namely Cys-503 and Cys-506, are located in the IRE binding domain(56) .

It must be recalled that some NO synthase product(s) can alter IRP in living cells sufficiently to change both its functions reciprocally(7, 9) . The exact nature of the NO synthase product which metamorphoses IRP into IRE-binding protein is still open to question. Whether NO itself is this molecule is controversial(7, 12, 17) , but this issue was not addressed here. However, according to the results of our experiments with NO donors (performed here in aerobiosis), we feel justified in proposing that the right ``track'' to follow in order to identify the molecule responsible for NO synthase-dependent conversion of IRP into IRE-binding protein, is probably to look for nitrosating species resulting from the reaction between NO and oxygen (NOx, NxOy, etc.) (57) and to determine their adequate redox environment. Finally, the differential effects of ROS and NO (or NO-oxygenated products) on IRP imply that the latter is a biosensor able to discriminate between each of these classes of reactive molecule.

In conclusion, if the results of the in vitro manipulations performed in our study indeed reflect the in vivo situation, our data support the assumption that neither O(2) nor ONOO is per se the physiological molecule which converts IRP from its aconitase form into its IRE binding form. In contrast, as several NO-releasing products promoted IRE binding in cell cytosol, we conclude that NO, or certain oxidized or redox-activated species derived from NO but different from ONOO, are the most likely molecules responsible for modulating IRP functions in living cells expressing NO synthase.


FOOTNOTES

*
This work was supported by a grant from the Association pour la Recherche sur le Cancer (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: U 365 INSERM, Section de Recherche, Institut Curie, 26, rue d'Ulm, 75231 Paris Cedex 05, France. Tel.: 33-1-4234-6713; Fax: 33-1-4407-07 85.

(^1)
The abbreviations used are: IRP, iron regulatory protein; IRE, iron-responsive element; GSH, glutathione; GSNO, S-nitrosoglutathione; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline: ROS, reactive oxygen species, SNC, S-nitrosocysteamine; SOD, superoxide dismutase; sper/NO, spermine/NONOate.


ACKNOWLEDGEMENTS

We thank Dr. Lukas Kühn for helpful discussion and for providing the plasmid pSPT-fer, and Prof. Marc Fontecave for providing S-nitrosocysteamine. The secretarial assistance of A. Birot is gratefully acknowledged.


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