Iron Regulatory Protein 2 as Iron Sensor

IRON-DEPENDENT OXIDATIVE MODIFICATION OF CYSTEINE*

Dae-Kyung KangDagger §, Jinsook JeongDagger §, Steven K. Drake||, Nancy B. WehrDagger , Tracey A. Rouault||, and Rodney L. LevineDagger **

From the Dagger  Laboratory of Biochemistry, NHLBI, National Institutes of Health and the || Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, January 20, 2003, and in revised form, February 11, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iron metabolism is exquisitely regulated by all organisms, from bacteria to humans. In mammals, the iron regulatory proteins (IRPs)1 mediate the coordinate expression of proteins that participate in iron metabolism (1, 2). When iron stores are low, the IRPs bind to an RNA stem-loop structure known as an iron-responsive element (IRE) located in either the 5'- or 3'-untranslated region of mRNA. If the IRE is close to the cap site, binding of the IRP blocks initiation of translation, causing a decrease in the level of the protein encoded by that mRNA. Conversely, when the IRE is located in the 3'-untranslated region of the transferrin receptor transcript, binding of the IRP stabilizes the mRNA by decreasing susceptibility to nuclease attack, causing an increase in the level of the protein encoded by the mRNA (3).

Mammals have two known IRPs, IRP1 and IRP2, with the tissue roles of each still being defined. IRP1 contains an iron-sulfur center, whereas IRP2 does not. The general mechanism by which each IRP is regulated is distinct (1). When cellular iron stores are low, IRP1 lacks a functional iron-sulfur center and binds to its IRE targets. When iron stores are sufficient, IRP1 regains its full iron-sulfur center, loses the ability to bind to IRE, and functions as a cytosolic aconitase. The cellular levels of IRP1 are unaffected by iron status in most cell types.

In contrast, IRP2 protein and IRE binding activity are readily detected when iron stores are limited but are low or absent when iron stores are sufficient (4, 5). The decrease in IRP2 protein occurs as a consequence of rapid degradation by the proteasome; synthesis of the protein is constitutive and does not vary substantially with iron status. The sequences of the two IRPs are similar except that IRP2 contains one domain not present in IRP1, and this domain is both required and sufficient to confer susceptibility to iron-dependent degradation. Deletion of the degradation domain produces an IRP2 whose levels no longer vary with iron status. Conversely, insertion of the degradation domain into IRP1 renders it susceptible to iron-triggered degradation (6).

We showed previously that IRP2 is oxidatively modified and ubiquitinylated in vivo, after which it is degraded by the proteasome (6). The oxidation requires oxygen and leads to the introduction of carbonyl groups into the protein, a hallmark of site-specific, metal-catalyzed oxidations (7). The oxidative modification of IRP2 is also mediated in vitro by a model metal-catalyzed oxidation system consisting simply of a reducing agent (dithiothreitol (DTT)), oxygen, and a redox-cycling cation (iron) (6). The in vitro oxidatively modified IRP2 is a substrate for ubiquitinylation, whereas the native form is not.

Oxidative modifications are capable of converting native proteins from proteasome-resistant to proteasome-sensitive substrates. For example, hypoxia-inducible factor-1 is oxidized at prolines to generate hydroxyproline (8-10). The reaction is mediated by an iron- and oxygen-dependent prolyl hydroxylase that functions as an oxygen sensor. Metal-catalyzed oxidation of proteins may also occur non-enzymically, and the modified proteins are usually much more susceptible to digestion by the proteasome (11). In metal-catalyzed oxidation, the residue that is modified lies at or very near a binding site for the divalent cation, supporting site-specific generation of a reactive oxidizing species (12). All amino acids are susceptible to metal-catalyzed oxidation of their side chains, but histidine, arginine, lysine, and proline are particularly sensitive. We hypothesized that the degradation domain of IRP2 contains an iron-binding site that has evolved to mediate its oxidative modification when both iron and oxygen are present. We found that this was the case and that the covalently modified residue was a cysteine. Oxidation generated multiple products, including dehydrocysteine and aminomalonic acid, the shorter homologue of aspartic acid.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of IRP2 Protein and Peptides-- Full-length IRP2 with a C-terminal Myc tag was expressed in the yeast Pichia pastoris and purified with an RNA affinity column as described (13). The coding sequence for the 63-residue peptide (P138-200) was obtained by PCR using 5'-ggagaattccatatggcaatacagaatgcaccaaatcc-3' as the forward primer and 5'-cgaggatccctacaggatgggtgtattctcaatc-3' as the reverse primer. It was inserted into the prokaryotic expression vector pET17b (Novagen, Madison, WI) at the NdeI and BamHI sites. Site-specific mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All three single cysteine to alanine mutants were constructed, C168A, C174A, and C178A. Then all three combinations of double mutants were constructed along with the triple mutant with the three cysteines changed to alanines. Wild-type or mutant plasmids were transformed into Escherichia coli XL1-Blue supercompetent cells (Stratagene). Each construct was sequenced to confirm that the desired sequence was obtained.

For expression the constructs were transformed into E. coli BL21(DE3) (Novagen) and induced by 0.4 mM isopropyl-beta -D-thiogalactopyranoside at 37 °C for 3 h. Cells were harvested by centrifugation at 17,000 × g for 15 min. Cells were disrupted by sonication, and debris was removed by centrifugation, again at 17,000 × g for 15 min. The peptide was concentrated and partially purified by precipitation with 70% ammonium sulfate. The pellet was dissolved in 1 M ammonium sulfate and centrifuged again. The supernatant was applied to a phenyl-5PW column, 0.75 × 7.5 cm (Tosoh Biosep, Montgomeryville, PA), which was equilibrated with 50 mM sodium acetate, pH 5.2, M ammonium sulfate. A decreasing ammonium sulfate gradient, 1 to 0 M, eluted the bound material. Fractions containing the IRP2 peptide were identified by 18% acrylamide SDS-PAGE electrophoresis or MALDI-TOF mass spectrometry (Hewlett-Packard, Palo Alto, CA). These were applied to a C18 reverse-phase column (Vydac 218TP5205, Hesperia, CA) equilibrated with 0.05% trifluoroacetic acid. Elution was accomplished with a linear gradient of acetonitrile, 0.05% trifluoroacetic acid, increased at 1%/min. Fractions containing the recombinant peptide were pooled, dried by vacuum centrifugation (Savant SpeedVac, Holbrook, NY), dissolved in 50 mM Hepes, 6 M guanidine HCl, 2 mM Tris(2-carboxyethyl)phosphine, pH 7.2, and then dialyzed against 50 mM Hepes, pH 7.2. Aliquots were stored at -20 °C. Masses of the wild-type and mutant peptides were measured by electrospray mass spectrometry (Agilent 1100 series HPLC-MSD, Palo Alto, CA). Measured masses were within 0.6 atomic mass units of that calculated from the sequence. We also determined the cysteine content by amino acid analysis of the alkylated peptide with and without treatment with DTT. These were the same, confirming that the cysteines were fully reduced.

Oxidative Modification and Analyses-- As in our earlier study (6), full-length IRP2 or recombinant peptide was oxidatively modified by exposure to a model metal-catalyzed oxidation system consisting of iron, DTT, and oxygen from air. Except when indicated otherwise, 30 µM peptide was incubated at 37 °C in 50 mM Hepes, pH 7.2, in room air with 10 mM DTT and 5 µM ferric chloride. The reaction was stopped by the addition of the chelator diethylenetriamine pentaacetic acid to 2 mM. Anaerobic experiments were conducted in an anaerobic chamber (Coy Laboratory Products 7150-000, Grass Lake, MI) in an atmosphere of 97% nitrogen, 3% hydrogen. Oxygen content was <= 1ppm.

Protein or peptide were alkylated by making the solution 6 M in guanidine HCl (by the addition of 1 gm/ml solid), 5 mM DTT, 100 mM Tris, pH 8.5, and 1 mM EDTA. Incubation at 37 °C for 30 min assured reduction of disulfides, and then iodoacetamide was added to a final concentration of 20 mM. After 30 min of additional incubation, excess alkylating agent was scavenged by adding an additional 10 mM DTT and allowing the solution to stand for 5 min. If the protein or peptide was not alkylated, reduction of thiols was accomplished by incubation with 2 mM Tris(2-carboxyethyl)phosphine at 37 °C for 30 min followed by acidification with trifluoroacetic acid to pH 2-3 before HPLC-mass spectral analysis.

Peptides were alkylated and then desalted with C18 ZipTips (Millipore, Bedford, MA) before amino acid analysis. Alkaline hydrolysis in 5 M NaOH, 3% thiodiglycol was carried out at 155 °C for 45 min (14). After cooling, the solution was neutralized with 25% acetic acid. Acid hydrolysis and amino acid analyses were performed as described (15). Carboxyamidomethylcysteine was converted to carboxymethylcysteine during the acid hydrolysis, and the latter is known to be partially degraded during hydrolysis. The average loss in 30 separate analyses of native peptide was 21%, similar to the 15% that we measured for the cysteines in the proteins lysozyme and glutamine synthetase. To correct for the loss, we normalized the value obtained with native peptide to the expected value of 3.0. The measured mass of the native, alkylated peptide was equal to that calculated from its sequence, confirming that three cysteines were present per peptide molecule.

Lys-C (Wako, Richmond, VA) digestion was performed at 37 °C in 50-100 mM Tris, pH 8.5, in the presence of 0.5-1.0 mM EDTA using a 1:40-1:50 ratio of enzyme to protein or peptide. The reaction was stopped by acidification with trifluoroacetic acid. Before application to the reverse phase column (Vydac 218TP5205, Hesperia, CA), the solution was made 6 M in guanidine HCl by the addition of 1 g/ml solid salt. To avoid suppression of the mass spectrometer by the guanidine, the sample was applied to the column with an HPLC pump separate from the analytical HPLC-mass spectrometer. Chromatographic separation and electrospray mass spectrometry were performed as previously described (16). Mass spectra were deconvoluted with the software provided by the instrument manufacturer (Agilent Technologies, Palo Alto, CA, Chemstation Version 9). Total recovery of recombinant peptide and products was calculated from the area of the chromatogram at 210 nm, and recovery of the native form was calculated from its absolute abundance in the total ion chromatogram.

For sequencing by tandem mass spectrometry, full-length IRP2 was allowed to undergo oxidative modification at 4 °C, alkylated, and then digested with endoproteinase Lys-C. The loop peptide was purified by reverse phase chromatography and then digested with trypsin, with the progress monitored by MALDI-TOF mass spectrometry. Digestion conditions were the same as for Lys-C except that 1 mM ammonium carbonate replaced Tris/EDTA as the buffer to facilitate the MALDI-TOF monitoring. When intermediate peptides appeared, the reaction was stopped by acidification, and the resultant peptides were separated by reverse phase chromatography as described above. The purified peptides were subjected to sequencing by tandem mass spectroscopy on a Micromass QTOF2 mass spectrometer equipped with their CapLC (Waters, Milford, MA). Data were processed with the Micromass MassLynx and MaxEnt software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Degradation Domain Is Oxidatively Modified-- We showed earlier that a model metal-catalyzed oxidation system (iron/DTT/oxygen) was capable of oxidatively modifying the full-length IRP2, rendering it susceptible to ubiquitinylation (6). We have now utilized this model system in studies aimed at determining the site of oxidative modification. The ~110,000-Da protein was digested with endoproteinase Lys-C to generate a peptide collection for mapping by reverse phase HPLC and mass spectroscopy. In a protein as large as IRP2, covalent modifications may be difficult to detect because of the complexity of its peptide map. However, the UV chromatogram did reveal one peak that consistently changed upon exposure to the complete iron oxidizing system (Fig. 1), but not if iron or oxygen were omitted nor if the chelator DPTA was added. The mass spectrum of the peak from non-oxidized protein showed a peptide of mass 5629.8, identifying it as peptide P165-216 (calculated mass = 5630.4), overlapping part of the 73-residue degradation domain encompassing residues 137-209 (5). Edman sequencing confirmed the identification. The gradient for the chromatogram in Fig. 1 was optimized to separate this degradation domain peptide from others, although it still co-eluted with P697-714 (measured mass = 2173.1; calculated = 2173.5). Nevertheless, the peptide map of IRP2 exposed to 1 µM iron, 10 mM DTT, air clearly showed a shoulder that eluted earlier than the main peak. The mass of the peptide in the shoulder was 5627.7, whereas those in the main peak were 5629.7 and 2173.1. 


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Fig. 1.   Lys-C peptide map of IRP2. The upper panel is the map of the control IRP2, and the lower panel is that which was exposed to metal-catalyzed oxidation for 30 min in room air with 1 µM FeCl3, 10 mM DTT and an IRP2 concentration of 0.1 mg/ml. The eluting gradient was optimized to separate the degradation domain peptide (P165-216) from others, as described under "Experimental Procedures." It eluted just after 40 min. The arrow in the lower panel shows the leading shoulder containing a peptide whose mass was 2 atomic mass units less than the native. This difference was reproducibly observed, whereas others such as the changes just after 35 min were not.

Thus, the model metal-catalyzed oxidation system induced a covalent modification with a mass decrease of 2 atomic mass units. This was not due to disulfide formation because the Lys-C peptide digest was reduced with Tris(2-carboxyethyl)phosphine or DTT before injection into the HPLC. When cysteine is alkylated with iodoacetamide the resulting carboxyamidomethylcysteine gains 57 atomic mass units. Alkylation of IRP2 should convert the degradation peptide P165-216 from mass 5630.4 to mass 5858.6 because it contains 4 cysteines. The measured mass of the native IRP2 was 5858.2, whereas that of the modified peptide was 5799.3. This is 59 atomic mass units less than the native peptide. This deficit implies that the oxidized residue can no longer be alkylated by iodoacetamide.

Further characterization of the iron-dependent modification was hampered by the difficulty of obtaining sufficient material from the full-length IRP2. Increasing the concentration of iron or the time of exposure to the oxidizing system led to poor recovery of the degradation peptide, perhaps because of aggregation of the oxidized products (see "Discussion"). In addition, the peptide of interest constitutes only 5% of the total IRP2.

A Peptide from the Degradation Domain Is Also Susceptible to Oxidative Modification-- We therefore produced a recombinant peptide with the sequence of residues 138-200 from the degradation domain, including the 3 cysteine residues in the middle of the domain. It was purified to homogeneity as assessed by SDS gel electrophoresis, reverse phase chromatography, and both MALDI-TOF and electrospray mass spectrometry. Exposure of this peptide to the iron/DTT/oxygen model system induced the same changes as observed in the full-length IRP2. Recovery of material was typically 93-95% in one major peak and ~2% in a small peak eluting 2-3 min before the major peak. Before exposure to the oxidizing system the mass spectrum was homogeneous with the mass that of the native peptide. After exposure, the mass spectrum became heterogeneous, containing both residual native peptide and products. The most prominent product, representing ~5% of the starting material, was again 59 atomic mass units less than the native peptide when alkylated (native = 6694 atomic mass units; modified = 6635 atomic mass units) and 1-2 atomic mass units less when not alkylated.2 As with the full-length protein, these results indicate that oxidative modification converts a cysteine to a product that is 2 atomic mass units less than cysteine and which can no longer be alkylated by iodoacetamide.

Although the mass spectrum became heterogeneous, the native form was always readily deconvoluted so that we could determine the fraction of the peptide that was still native. After a 2-h exposure to the oxidizing system, virtually all of the peptide was modified. Amino acid analysis of this and the native peptide confirmed the loss of one cysteine; no other modifications were detected (Table I). Loss of the cysteine residue required only micromolar concentrations of iron (Table II). Examination of the time course of the oxidation revealed that loss of the native mass coincided with loss of the cysteine residue (Fig. 2). Neither loss of cysteine nor change in mass occurred if the incubation was performed anaerobically nor if iron was chelated by diethylenetriaminepentaacetic acid (see Table IV). Thus, the IRP2 degradation domain is susceptible to iron and oxygen-dependent oxidative modification of a cysteine residue.


                              
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Table I
Amino acid analysis of recombinant peptide
We analyzed the results by ratio normalization as described (11). The 95% limits are defined by the average ±2 S.D. Only the Cys content of the oxidized peptide was significantly altered. The control and oxidized results (3-h exposure) are the average of 2 separate experiments. Cysteine was normalized to 3.0 in the control as described under "Experimental Procedures."


                              
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Table II
Effect of iron concentration on loss of cysteine residue
Recombinant peptide was exposed to the metal-catalyzed oxidation system for 100 min in room air. The DTT concentration was 10 mM. Cysteine content was determined by amino acid analysis, and the value shown is the average of two experiments.


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Fig. 2.   Time course of oxidative modification of the recombinant degradation domain peptide. After alkylation the samples were divided for determination of peptide mass by HPLC-mass spectrometry () and for cysteine residues (black-square) by amino acid analysis after acid hydrolysis. The metal catalyzed oxidation system was 5 µM iron and 10 mM DTT in air.

The efficiency of direct oxidation of cysteine was high, as shown in Fig. 2. However, close examination of the chromatogram of the peptide exposed to the iron-oxidation system revealed a small peak eluting ~3 min earlier than the major peak, whose area was ~2% of the area of the major peak. Although this peak was thus barely visible in the UV chromatogram, the mass spectrometer readily established the identity of peptides in the peak (Table III). They resulted from cleavage of the parent peptide on the amino side of each cysteine. This observation establishes that the three cysteine residues are located at the site of free-radical generation, and thus, likely participate in the binding of iron. In addition to cleavage at each cysteine residue, the peptide bonds at Lys-160 and Lys-165 were also susceptible to hydrolysis, indicating that they are also at or near the iron-binding site. In the course of purifying IRP2 from rat liver, Guo et al. (17) noted the presence of two forms, ~104-kDa full-length and 83 kDa. The lower molecular mass form was sequenced and found be the product of clipping between Ser-192 and Gln-193, which we have not yet observed in our studies of the recombinant peptide.


                              
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Table III
Peptides formed by oxidative cleavage
Masses were determined by deconvolution of the spectra from the total ion chromatogram.

For cleavage at each of the three cysteine residues, the masses of the product peptides were 1 atomic mass unit less than would occur with simple peptide bond hydrolysis. This is the expected result for peptide bond cleavage by reactive oxygen species after the alpha -amidation mechanism (18), likely proceeding through formation of dehydrocysteine followed by peptide bond cleavage (19, 20). Cleavage at the lysines gave peptides with masses expected for peptide bond hydrolysis, indicating cleavage via a mechanism other than alpha -amidation (10).

The model metal-catalyzed oxidation system consists of micromolar concentrations of iron, oxygen from air, and millimolar concentrations of DTT. We investigated whether all were necessary for oxidation of the peptide (Table IV). Oxygen and iron are required for the reaction. DTT is not required, although its presence stimulated the reaction.


                              
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Table IV
Requirements for oxidation of the peptide
The complete metal-catalyzed oxidation system contained 5 µM iron, 10 mM DTT and was exposed to room air. The chelator DPTA (1 mM) was used to test the requirement for iron, and incubation in an anaerobic hood tested the requirement for oxygen. The time course was followed over 4 h and established that changes reached a plateau by ~150 min. The loss of cysteine shown below is the average of the 150- and 200-min time points.

Role of Each Cysteine-- To assess the function of the cysteine residues, we constructed site-specific mutants in which all combinations of one, two, or all three cysteine residues were changed to alanine. The seven mutants were tested for susceptibility to iron-mediated modification. Mutation of 1 cysteine decreased the rate of oxidative modification by over 50%, and mutation of 2 cysteines decreased the rate even further (Fig. 3). There was no detectable difference in susceptibility among the single mutants nor the double mutants, again consistent with the suggestion that all three cysteine residues chelate the iron cation. The triple mutant has no cysteine residues, and only the native mass was detected in the main peak after exposure to the metal-catalyzed oxidation system. However, ~1% iron-dependent peptide bond cleavage could still be observed. The cleavage sites included Lys-160 and Lys-165, as in the wild-type peptide (Table III). We occasionally observed cleavage at other residues as well, most commonly between Phe-190 and Ser-191.


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Fig. 3.   Effect of the number of cysteine residues on oxidative modification. The wild type and all six site-specific mutants (Cys right-arrow Ala) were exposed to metal-catalyzed oxidation for 2 h, and the loss of cysteine was determined by amino acid analysis. The results for the three mutants with one cysteine converted to alanine were the same as were those for the three mutants with two cysteines converted to alanine. Thus, the averages are shown. Note that although the wild-type sequence has three cysteine residues, a maximum of one can be modified (Fig. 2).

Dehydrocysteine Formation from Cysteine-- We hypothesized that the initial step in the oxidative modification would be conversion of cysteine to dehydrocysteine (Fig. 4), which incidentally is also 2 atomic mass units less than cysteine. The alkene form of dehydrocysteine is a thiol, which would be alkylated by iodoacetamide (21). When we followed the time course of a tryptic digestion of the alkylated, oxidatively modified peptide, we detected partially digested peptides that were 2 atomic mass units less than the mass calculated for the native peptide. These were not seen when the digestion went to completion, generating relatively low molecular weight final products. This observation suggested that dehydrocysteine might be relatively stable in a larger peptide but not in smaller ones. We therefore performed a partial tryptic digestion of the alkylated, oxidatively modified peptide. Products were separated by reverse phase chromatography and then subjected to tandem mass spectroscopy for sequence analysis. We identified and sequenced native and oxidatively modified peptide containing residues 176-185. The measured monoisotopic mass of the native peptide was 1036.46 atomic mass units and that of the modified peptide was 1034.44 atomic mass units (native calculated = 1036.42). Analysis of the native peptide sequence showed a residue of 160 atomic mass units at the position where cysteine was expected but 158 atomic mass units for the modified peptide (Fig. 5). The carboxyamidomethyl group of alkylated cysteine or dehydrocysteine can be cleaved in the mass spectrometer to generate dehydroalanine. If derived from cysteine the mass change is -91 atomic mass units and, if derived from dehydrocysteine, it is -89 atomic mass units, as observed in the lower panel of Fig. 5. Sequencing of other peptides from the same digest demonstrated dehydrocysteine was formed at each of the three cysteine positions, Cys-168, Cys-174, and Cys-178.


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Fig. 4.   Proposed pathway for oxidation of cysteine.


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Fig. 5.   Tandem mass spectrometric sequencing of P176-185. The sequence of P176-185 is GSCDSGELGR. The upper panel shows the spectrum for the native peptide, and the lower panel shows the spectrum for the oxidatively modified peptide. The mass differences and corresponding residue are shown for the y ion series. The only difference between the native and oxidatively modified peptide is the conversion of Cys-178 to dehydrocysteine. Thus, fragments of mass 733 or less were the same mass in both peptides, whereas a 2-atomic mass unit (amu) difference was observed for the larger fragments.

Aminomalonic Acid Is a Product-- As noted above, the mass of one of the products of cysteine oxidation was 2 atomic mass units less than the native peptide and could not be alkylated by iodoacetamide. These characteristics would fit thiohistidine, formed by the covalent linkage of a cysteine and histidine residue, but the recombinant peptide does not have any histidine residues. Not surprisingly then, it was not detected in the hydrolysate of the oxidatively modified peptide (authentic thiohistidine eluted just after serine in our amino acid analysis system). Aminomalonic acid is a dicarboxylic acid one methylene group shorter than aspartic acid. Its mass is 119 Da, 2 atomic mass units less than cysteine, and it is not alkylated by iodoacetamide. It readily decarboxylates to yield glycine upon acid hydrolysis (22), and we also observed decarboxylation of a synthetic 9-residue peptide containing aminomalonic acid when subjected to MALDI-TOF mass spectroscopy.3 However, aminomalonate is relatively stable to alkaline hydrolysis (23). Authentic aminomalonic acid elutes as a shoulder after aspartic acid in our system, and it was detected in the alkaline hydrolysate of the oxidatively modified peptide (Fig. 6).


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Fig. 6.   Aminomalonic acid is present in the oxidatively modified peptide. These are chromatograms from amino acid analysis after alkaline hydrolysis. Aspartate is the first eluting amino acid, at about 4.4 min, whereas aminomalonate elutes slightly later. The control, native peptide is in panel D, and the oxidatively modified peptide is in panel C. Authentic aminomalonate (Ama) is shown in panel A, and the mixture of aminomalonate and oxidatively modified peptide is in panel B, demonstrating coelution.

Aminomalonic acid has been detected in proteins isolated from E. coli and from human atherosclerotic plaques (22). The original investigators noted that the aminomalonate could be derived via beta -elimination of sulfur from cysteine (23), a point that we have now confirmed experimentally. The conversion of cysteine to the acidic aminomalonate could induce conformational changes that are important in the recognition of IRP2 for ubiquitinylation and degradation by the proteasome.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IRP2 regulates iron metabolism, and IRP2 in turn is regulated by iron. We have demonstrated that the degradation domain forms an iron-binding site that readily undergoes iron-catalyzed oxidative modification. The oxidative modification proceeds efficiently with a non-enzymatic system consisting of oxygen, iron, and a reducing agent. However, iron metabolism is exquisitely regulated in vivo, so it would not be surprising to find that other factors, presumably proteins, modulate the reaction. For example, a protein could bind to the degradation domain and shield it from oxidative modification even when cellular iron pools are relatively replete. There may also exist an IRP2 oxidase that speeds the oxidation reaction and which is itself subject to regulatory control. If so it would provide a mechanism similar to the prolyl hydroxylase that triggers the degradation of hypoxia-inducible factor-1 (8, 9). When cellular oxygen is sufficient, hypoxia-inducible factor is oxidatively modified by an iron-dependent prolyl hydroxylase that converts two proline residues to hydroxyprolines (10). The covalently modified protein then interacts with a ubiquitin ligase complex becomes polyubiquinylated and is degraded by the proteasome.

However, the analyses presented in this paper establish that the degradation domain can function as a "self-sufficient" iron sensor. It does not require the action of other proteins to become covalently modified in the presence of iron. It has an iron-binding site that facilitates interaction with oxygen to generate a reactive oxygen species which covalently modifies a cysteine residue. All three central cysteines participate in forming the site, and mutation of any one of them to an alanine substantially decreases the rate of oxidation of the remaining two. The details of the structure with and without bound metal will require direct determination by NMR or x-ray spectroscopy. However, the degradation domain is rich in basic residues, and both Cys-168 and Cys-174 are adjacent to arginines; Lys-160 and Lys-165 must be near the iron-binding site because their peptide bonds are occasionally cleaved during the oxidation process. These basic residues may serve to lower the pKa of the cysteine residues, allowing their thiolate anions to ligate the iron cation (Fig. 7).


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Fig. 7.   Schematized model of the iron-binding site of the degradation domain. The peptide bonds of the shaded residues were susceptible to iron dependent oxidative cleavage and are, thus, placed close to the iron atom.

The model metal-catalyzed oxidation system consists of micromolar concentrations of iron, oxygen from air, and millimolar concentrations of DTT. The DTT is included to assure reduction of ferric iron to the ferrous form. When we examined the requirements for oxidation of the peptide we found that oxygen and iron were required (Table IV). However, DTT was not required, although its presence stimulated the reaction. Because DTT is not required, it is likely that one of the cysteine residues in the degradation domain reduces bound ferric iron to the ferrous form. Molecular oxygen then binds to the site and reacts to convert one of the cysteine residues to dehydrocysteine. The dehydrocysteine may be stabilized within the IRP2 protein but more likely undergoes further reactions to yield multiple products, including aminomalonic acid (Fig. 4). Conversion of one cysteine to aminomalonate and other products may interfere with binding of iron to the remaining cysteine residues, explaining why only one cysteine is oxidized per molecule (Fig. 2).

Dehydrocysteine undergoes tautomerization analogous to the keto-enol tautomers of oxygen analogues (24). Consequently, it has chemical characteristics of both an alkene and a thioketone. The latter should react with carbonyl reagents such as 2,4-dinitrophenylhydrazine to yield the 2,4-dinitrophenylhydrazone and sulfide. IRP2 oxidatively modified in vivo or in vitro forms a 2,4-dinitrophenylhydrazone, which we had previously assumed to derive from an oxygen-containing carbonyl group, but one should now also consider that it was formed from the thioketone form of dehydrocysteine.

We do not yet know the details of the mechanism by which the cysteine is oxidized, but it may be similar to that utilized by isopenicillin-N synthase (25). This non-heme iron-dependent enzyme catalyzes the reaction of molecular oxygen with cysteine in the tripeptide L-aminoadipoyl-L-Cys-D-Val to form a dehydrocysteine. The proposed mechanism begins with the ligation of the thiolate of the cysteine to the ferrous iron center of the enzyme, creating an open iron coordination site to which oxygen binds. Then iron-dioxygen and the resultant iron-oxo species abstract hydrogens from the substrate without participation of residues of the enzyme. For IRP2 the first step would be the binding of ferric iron, which would be reduced by a cysteine to the ferrous state, allowing the binding of molecular oxygen. Abstraction of a second hydrogen would yield dehydrocysteine and a ferric hydroperoxy or ferryl moiety, which could further oxidize the dehydrocysteine. Ferric ion would be released to bind to another IRP2 molecule and catalyze another oxidation cycle. The ferric hydroperoxy intermediate might also simply dissociate (26), yielding ferric iron and an overall reaction of cysteine right-arrow dehydrocysteine + hydrogen peroxide.

As noted above, several lysine residues are at or close to the iron-binding site. Lysine residues are particularly sensitive to metal-catalyzed oxidation, with the major product being aminoadipylsemialdehyde (27). Stadtman (27) suggests one mechanism by which lysine could be oxidized, and it is possible that this is relevant to the oxidation of IRP-2. The epsilon amino group of the lysine is liganded to the iron atom, leading to the formation of a carbon-centered radical that then forms an epsilon - imido group. Reaction with water would yield ammonia and the aminoadipylsemialdehyde. However, in IRP2 the lysyl radical could abstract a hydrogen from a cysteine. This would regenerate the lysine while creating a cysteinyl radical, which could then be further oxidized. Thus, lysine residues may be direct participants in the reaction mechanism even though they are not modified in the final product.

Although the details of the oxidation mechanism of IRP2 remain to be experimentally elucidated, it is clear that the degradation domain has evolved to become an efficient iron sensor. It contains an iron-binding site that mediates reaction with oxygen to cause covalent modification of the site. This modification causes a structural change that may be recognized by the proteasome system which then degrades the oxidatively modified IRP2, a hypothesis that can now be tested in vivo. The degradation domain has thus harnessed a reactive oxygen generating reaction to provide an elegant means of regulating the susceptibility of IRP2 to proteolytic degradation. IRP2 could, therefore, function not only as a regulator of translation of mRNAs but also as a cellular iron sensor.

    Acknowlegments

We thank Wolff Kirsch and Tad Koch for advice and helpful discussions about aminomalonic acid. Earl Stadtman generously offered insightful suggestions during many discussions. Henry Fales and Lewis Pannell kindly provided access to the QTOF mass spectrometer.

    FOOTNOTES

* 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 contributed equally to this work.

Current address: 374-4 Easy Bio System, Uiwang City 437-020, Republic of Korea.

** To whom correspondence and requests for reprints should be addressed (E-mail preferred): NIH, Bldg. 50, Rm. 2351, Bethesda, MD 20892-0812. Tel.: 301-496-2310; Fax: 301-496-0599; E-mail: rlevine@nih.gov.

Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M300616200

2 With the recombinant peptide the native and oxidatively modified forms coelute from the HPLC whether alkylated or not. The mass resolution of our mass spectrometer is not sufficient to reproducibly resolve the two peptides when not alkylated (native = 6523.4; modified = 6521.4) so that we usually observed a single, weighted average of the two masses.

3 As in the case of dehydrocysteine-containing peptides, larger peptides containing aminomalonate were more stable to MALDI-TOF. For example, when the full-length (63-residue peptide (P138-200)) oxidatively modified peptide was analyzed by MALDI-TOF, we readily detected both the residual native peptide and the -59 atomic mass units peptide containing aminomalonate. Had the aminomalonic acid decarboxylated to yield glycine, the mass difference would have been -103. In contrast, the nine-residue peptide (P169-177, RGQTTBRGS) with cysteine replaced by aminomalonate (B) decarboxylated to glycine during MALDI-TOF analysis. We also used this peptide to confirm experimentally that aminomalonate is not alkylated by iodoacetamide.

    ABBREVIATIONS

The abbreviations used are: IRP, iron regulatory protein; IRE, iron-responsive element; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; P, peptide; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rouault, T., and Klausner, R. (1997) Curr. Top. Cell. Regul. 35, 1-19[Medline] [Order article via Infotrieve]
2. Hentze, M. W., and Kuhn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182[Abstract/Free Full Text]
3. Binder, R., Horowitz, J. A., Basilion, J. P., Koeller, D. M., Klausner, R. D., and Harford, J. B. (1994) EMBO J. 13, 1969-1980[Abstract]
4. Guo, B., Phillips, J. D., Yu, Y., and Leibold, E. A. (1995) J. Biol. Chem. 270, 21645-21651[Abstract/Free Full Text]
5. Iwai, K., Klausner, R. D., and Rouault, T. A. (1995) EMBO J. 14, 5350-5357[Abstract]
6. Iwai, K., Drake, S. K., Wehr, N. B., Weissman, A. M., LaVaute, T., Minato, N., Klausner, R. D., Levine, R. L., and Rouault, T. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4924-4928[Abstract/Free Full Text]
7. Levine, R. L. (2002) Free Radic. Biol. Med. 32, 790-796[CrossRef][Medline] [Order article via Infotrieve]
8. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) Science 292, 464-468[Abstract/Free Full Text]
9. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468-472[Abstract/Free Full Text]
10. Masson, N., Willam, C., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) EMBO J. 20, 5197-5206[Abstract/Free Full Text]
11. Rivett, A. J., and Levine, R. L. (1990) Arch. Biochem. Biophys. 278, 26-34[Medline] [Order article via Infotrieve]
12. Stadtman, E. R., and Levine, R. L. (2000) Ann. N. Y. Acad. Sci. 899, 191-208[Abstract/Free Full Text]
13. Allerson, C. R., Martinez, A., Yikilmaz, E., and Rouault, T. A. (2003) RNA (N. Y.) 9, 364-374
14. Levine, R. L. (1982) J. Chromatogr. 236, 499-502[CrossRef]
15. Reddy, V. Y., Desrochers, P. E., Pizzo, S. V., Gonias, S. L., Sahakian, J. A., Levine, R. L., and Weiss, S. J. (1994) J. Biol. Chem. 269, 4683-4691[Abstract/Free Full Text]
16. Taggart, C., Cervantes-Laurean, D., Kim, G., McElvaney, N. G., Wehr, N., Moss, J., and Levine, R. L. (2000) J. Biol. Chem. 275, 27258-27265[Abstract/Free Full Text]
17. Guo, B., Yu, Y., and Leibold, E. A. (1994) J. Biol. Chem. 269, 24252-24260[Abstract/Free Full Text]
18. Stadtman, E. R., and Berlett, B. S. (1999) in Reactive Oxygen Species in Biological Systems (Colton, C. , and Gilbert, D. J., eds) , pp. 657-675, Kluwer Academic/Plenum Publishers, New York
19. Patchornik, A., and Solar, S. (1964) J. Am. Chem. Soc. 86, 1206-1212
20. Patchornik, A., and Solar, S. (1964) J. Am. Chem. Soc. 86, 1212-1217
21. Baldwin, J. E., Bradley, M., Adlington, R. M., Norris, W. J., and Turner, N. J. (1991) Tetrahedron 47, 457-480[CrossRef]
22. Van Buskirk, J. J., Kirsch, W. M., Kleyer, D. L., Barkley, R. M., and Koch, T. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 722-725[Abstract]
23. Copley, S. D., Frank, E., Kirsch, W. M., and Koch, T. H. (1992) Anal. Biochem. 201, 152-157[Medline] [Order article via Infotrieve]
24. Okazaki, R. (1995) in Organosulfur Chemistry: Synthetic Aspects (Page, P., ed) , pp. 225-258, Academic Press, Ltd., London
25. Roach, P. L., Clifton, I. J., Hensgens, C. M., Shibata, N., Schofield, C. J., Hajdu, J., and Baldwin, J. E. (1997) Nature 387, 827-830[CrossRef][Medline] [Order article via Infotrieve]
26. Ortiz de Montellano, P. R., and De Voss, J. J. (2002) Nat. Prod. Rep. 19, 477-493[CrossRef][Medline] [Order article via Infotrieve]
27. Stadtman, E. R. (1990) Free Radic. Biol. Med. 9, 315-325[CrossRef][Medline] [Order article via Infotrieve]


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