From the 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
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ABSTRACT |
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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.
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.
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- 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
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.
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.
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.
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.
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
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.
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.
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 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).
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 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).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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, 1 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.
1ppm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
Amino acid analysis of recombinant peptide
Effect of iron concentration on loss of cysteine residue
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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 (
) by amino acid
analysis after acid hydrolysis. The metal catalyzed oxidation system
was 5 µM iron and 10 mM DTT in air.
Peptides formed by oxidative cleavage
-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
-amidation (10).
Requirements for oxidation of the peptide
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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 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).
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.
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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.
-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
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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 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 - 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.
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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.
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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.
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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.
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