Unformatted text preview: © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology PERSPECTIVE The role of iron regulatory proteins in mammalian iron
homeostasis and disease
Tracey A Rouault Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are mammalian
proteins that register cytosolic iron concentrations and posttranscriptionally regulate expression of iron metabolism genes
to optimize cellular iron availability. In iron-deficient cells, IRPs
bind to iron-responsive elements (IREs) found in the mRNAs
of ferritin, the transferrin receptor and other iron metabolism
transcripts, thereby enhancing iron uptake and decreasing
iron sequestration. IRP1 registers cytosolic iron status mainly
through an iron-sulfur switch mechanism, alternating between
an active cytosolic aconitase form with an iron-sulfur cluster
ligated to its active site and an apoprotein form that binds IREs.
Although IRP2 is homologous to IRP1, IRP2 activity is regulated
primarily by iron-dependent degradation through the ubiquitinproteasomal system in iron-replete cells. Targeted deletions
of IRP1 and IRP2 in animals have demonstrated that IRP2 is
the chief physiologic iron sensor. The physiological role of the
IRP-IRE system is illustrated by (i) hereditary hyperferritinemia
cataract syndrome, a human disease in which ferritin L-chain
IRE mutations interfere with IRP binding and appropriate
translational repression, and (ii) a syndrome of progressive
neurodegenerative disease and anemia that develops in adult
mice lacking IRP2. The early death of mouse embryos that lack
both IRP1 and IRP2 suggests a central role for IRP-mediated
regulation in cellular viability.
Iron is indispensable for the function of many prosthetic groups, including heme and iron-sulfur clusters, and animals have accordingly developed sophisticated systems to maintain iron homeostasis. Cellular iron
uptake, distribution and export must be tightly regulated, as insufficient
iron concentrations impair the function of numerous iron proteins,
whereas excess free iron can oxidize and damage the protein, nucleic
acid and lipid contents of cells. In humans, iron deficiency is the most
common cause of anemia in the world, and it interferes significantly
with normal cognitive development in children. Conversely, the iron
overload observed in common diseases such as hemochromatosis and
thalassemia causes liver and heart failure. Thus, organisms and individual cells must regulate iron metabolism to ensure that sufficient
iron is provided to supply heme, the iron-sulfur prosthetic groups of
Tracey A. Rouault is in the Cell Biology and Metabolism Branch, National
Institute of Child Health and Human Development, Building 18T, Room 101,
National Institutes of Health, Bethesda, Maryland 20892, USA.
e-mail: [email protected]
Published online 18 July 2006; doi:10.1038/nchembio807 406 mitochondrial respiratory-chain complexes, and cellular iron enzymes
such as ribonucleotide reductase1.
In mammals, dietary iron uptake across the duodenal mucosa is regulated through the expression of the intramembrane metal transporters
ferroportin and divalent metal transporter 1 (DMT1) in response to
signals from the liver1,2. After iron has traversed the intestinal mucosa, it
enters the bloodstream and binds tightly to transferrin3. Most cells can
effectively control iron uptake by regulating the amount of transferrin
receptor (TfR1)4 that is expressed on their plasma membranes. In addition, cells regulate expression of the cytosolic iron-sequestration protein
ferritin5 as well as numerous other proteins to optimize availability of
In mammalian cells, the IRPs (which are derived from a duplicated
gene pair) register metal availability mainly through direct interactions
with iron in the cytosol6,7. In iron-depleted cells, IRPs bind to IREs, RNA
elements within mRNAs that encode ferritin, transferrin receptor and
many other transcripts, and this IRP binding represses translation (Fig. 1a)
or prolongs mRNA half-life (Fig. 1b) depending on where the IRP binds
on the mRNA7,8. The IRP-IRE regulatory system enables cells to rapidly
adjust concentrations of available cytosolic iron and thereby optimize the
functioning of numerous iron-dependent cellular components.
IRPs bind to IREs in iron metabolism transcripts
IREs are highly conserved IRP binding sites that are found in the 5′
untranslated region (UTR) of transcripts that encode the H and L ferritin
subunits (H, highly expressed in heart; L, highly expressed in liver), in the
3′ UTR of TfR1 and in several other iron metabolism genes8. IRP1 and
IRP2 are ubiquitously expressed mammalian members of the aconitase
gene family9 that have adapted to sense cytosolic iron concentrations
and accordingly modify gene expression. Aconitases are enzymes found
in virtually all organisms that convert citrate to isocitrate through the
intermediate cis-aconitate in the citric acid cycle, the central cycle of
intermediary metabolism. In aconitases, a cubane iron-sulfur cluster is
ligated by three cysteines within the enzymatic active site cleft, and the
fourth iron of the cubane cluster binds substrate10. IRP1 and IRP2 are
highly related to the aconitase family, but only IRP1 has retained its ability
to function as an aconitase, whereas IRP2 apparently lost its aconitase
activity sometime during evolution6,7.
In cells that are depleted of iron, each IRP responds by binding to
IREs (Fig. 1). By binding to a single IRE located in the 5 UTR of an
mRNA, the IRP prevents translation of the mRNA (Fig. 1a), whereas
by binding to IREs in the 3′ UTR, the IRP protects the TfR transcript
from endonucleolytic cleavage and degradation (Fig. 1b). In cells that
are iron replete, IRPs do not bind IREs, and ferritin and other transcripts VOLUME 2 NUMBER 8 AUGUST 2006 NATURE CHEMICAL BIOLOGY © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology PERSPECTIVE
that have an IRE in the 5′ UTR are freely translated. Conversely, the TfR exporter ferroportin30. IREs have also been found in the 3′ UTR of
transcript undergoes cleavage by an uncharacterized endonuclease at a one isoform of the ferrous iron transporter, DMT1 (ref. 31), and in
specific site flanked by IREs in the 3′ UTR, and the cleavage products are mammalian glycolate oxidase32 (also known as hydroxyacid oxidase
2), but it is not yet clear whether these IREs contribute to regulation of
Acquisition of IRE binding ability by IRP1 may have occurred through expression of DMT1 (ref. 33) or glycolate oxidase34.
natural selection: accumulation of IRP1 lacking an iron-sulfur cluster in
iron-deficient cells could have allowed the apoprotein to bind to RNA IRE mutations cause hyperferritinemia and cataract syndrome
stem-loop structures of appropriate size and shape that appeared in the Members of families with mutations in the IRE of the ferritin L chain
highly mutable UTRs of some mRNAs. When expression from transcripts have high serum concentrations of ferritin and are prone to development
encoding iron metabolism proteins was favorably affected, IRP binding of early-onset cataracts. Since the initial description of the hereditary
could have resulted in selective retention of the
IRE within the transcript. Selection of IREs may
be an ongoing evolutionary process, given that
a Ferritin mRNA
One IRE in 5´ UTR
UTR sequences are not constrained by having
to encode protein.
IRE stem loops have conserved sequence
features, including a six-member loop in which
the sequence of the first five residues is usually
CAGUG, and upper and lower stems that are
separated from one another by an unpaired
cytosine on the 5′ side (Fig. 2)11,12. Fully functional IREs are likely to have longer lower stems
than those depicted here, perhaps to allow more
extensive interactions of the RNA with IREbinding proteins (that is, IRPs) or to stabilize
the conformation of the IRE stem loop13,14. Base
pairing between loop residues 1 and 5 of the IRE
IRE, occupied by IRP,
stabilizes the loop structure11, and in vitro bindinitiation
ing studies have shown that other residues may
functionally substitute for the C1-G5 base pair
of the loop if base pairing is maintained15,16.
IRE, unoccupied, allowing
The structure of the IRE seems to function
polysome formation and
as a ‘molecular ruler’ that preserves a specific
increased ferritin synthesis
distance and spatial orientation between IRE
residues that may directly and specifically contact IRE-binding proteins. Possible candidates
b TfR mRNA
Five IREs in 3´ UTR
for such specific contacts include the G3 of the
loop, which has an unusual syn conformation
in the NMR structure, and the bulge cytidine
that separates the upper and lower stems
though substitution of other residues in the
bulge position is most likely compatible with
high-affinity binding17, and it is possible that
IRP1 and IRP2 have individual specific targets
that vary from the consensus sequence15,16,18.
Ferritin IREs have a more complex bulge and
a lower stem, and they are often represented
with a three-residue bulge19,20, although data
16 and analysis of human
from selection studies
mutations14,21 strongly imply that the middle
residue of the proposed bulge forms a base pair
with a residue on the opposite side of the stem
One or more IREs, occupied
by IRP, protecting mRNA
After initial characterization of the IRE
IRE, unoccupied, rendering
step, mRNA degradation
mRNA susceptible to
stem-loop structure22,23, IREs have been found
in the 5′ UTRs of the erythocytic form of
aminolevulinic acid synthase (eALAS, which
Figure 1 Ferritin translation and TfR mRNA degradation are regulated by IRP binding. (a) In
catalyzes the first step in heme synthesis)24,25,
iron-depleted cells, IRP binding to the IRE in the 5′ UTR interferes with translational initiation.
in mammalian mitochondrial aconitase
(b) Binding of IRPs to IREs in the TfR 3′ UTR protects the transcript from endonucleolytic cleavage
in the succinate dehydrogenase b subunit of and degradation. The arrow marks the site at which an unknown endonuclease cleaves the TfR
Drosophila melanogaster29 and in the iron transcript in iron-replete cells when it is unprotected by IRP binding6. NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 8 AUGUST 2006 407 © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology PERSPECTIVE
hyperferritinemia cataract syndrome (HHCS)35, numerous familial
mutations have been described21,36, and the degree of hyperferritinemia
and cataract severity has been correlated with the degree to which the
mutation impairs IRP binding14. Because patients with HHCS have high
concentrations of ferritin in their blood, candidates for HHCS are easily
identified by blood tests, and many mutations of the ferritin L-chain IRE
have been described, some of which are depicted in Figure 3. Mutations
of the ferritin L-chain IRE confer a dominant phenotype, because IRP
binding to transcripts from the mutant allele is partially or completely
attenuated, and ferritin L-chain expression increases even though translation of the other ferritin L-chain allele is repressed normally. Thus,
HHCS illustrates the importance of ferritin translational repression in
normal physiology. according to the dimensions and shape determined in its NMR structure
solution)11. The crystal structure of the holoprotein shows a closed conformation that does not allow substrates to access the active site, unless
dynamic motion of the structure allows small interdomain shifts within
the holoprotein, as was previously predicted10. Notably, reseachers have
identified a partially connected network of hydrophilic cavities in the
crystal structure of IRP1 at the domain interfacial region, and Dupuy
et al. have proposed that these channel-like structural features may be
involved in guiding the movement of substrates and products to and from
the active site, perhaps aided by a small rotation of domain 4. The IRE
has a shape much like that of the portion of domain 4 that faces domains
1–3, thereby enabling the IRE to potentially substitute for domain 4 and
interact with the portions of domains 1–3 that form the active site cleft.
In addition, electrostatic modeling of domain 4 has revealed numerous
positive charges on the domain 4 surface that normally faces the active
site cleft, and these positively charged residues may bind to the negatively charged phosphate backbone of the IRE in the IRP-IRE complex.
Thus, one side of the IRE appears to mimic the size and conformational
features of domain 4, allowing it to substitute for domain 4 by forming
favorable contacts with domains 1–3, while on the opposite side of the
IRE, charge interactions may allow the IRE to bind to the displaced fourth
domain. Crystallization of the IRP-IRE complex60 and solution of the
crystal structure should allow a detailed characterization of the RNA
binding site of IRP1.
Both IRP1 and IRP2 bind IREs with high affinity in iron-depleted
cells, as demonstrated in many different cell lines by gel-shift assays that
use radiolabeled IRE to reveal binding activity. Efforts to elucidate the
mechanisms by which IRPs register cytosolic iron concentrations have
been ongoing since their initial characterization. The structure of IRP1 and formation of the IRP-IRE complex
In mammalian cells, IRP1 and IRP2 bind to IREs and modify expression
of ferritin, TfR and other transcripts in iron-depleted cells. Investigators
originally purified and cloned IRP1 by using its ability to bind IREs as
an assay37–40, and they were able to clone IRP2 simultaneously with IRP1
because of sequence homology37, although IRP2’s role as a second IREbinding protein was not recognized for several years41,42. IRP1 and IRP2
are derived from duplicated mammalian genes, and the human forms
are 56% identical to one another. IRP1 is a functional aconitase that
interconverts citrate and isocitrate in the cytosol43,44. This reaction is also
catalyzed in the mitochondrial matrix by mitochondrial aconitase, a key
enzyme of the citric acid cycle that is encoded by a separate gene45. IRP1
contains a [4Fe-4S] cluster that is ligated to the active site by binding of
Cys437, Cys503 and Cys506 to three iron atoms of the cluster, whereas
the fourth iron binds solvent and substrate10,44.
The recent solution of the crystal structure of IRP1 (ref. 46) reveals
that the structure of IRP1 is very similar to that of mitochondrial aconi- IRP1 has an iron-sulfur switch that determines its activity
tase47, as was predicted on the basis of sequence conservation between Researchers recognized a possible mechanism for regulation of the IREIRP1 and mitochondrial aconitase10. When IRP1 loses its iron-sulfur binding activity of IRP1 when it became apparent that IRP1 alternates
cluster, it acquires the ability to bind to IREs with high affinity, and between two major forms: the cytosolic aconitase form, which contains
when the cysteines that ligate the iron-sulfur cluster are mutagenized, a [4Fe-4S] cluster bound to the enzymatic active site and which does not
IRP1 becomes a constitutive IRE-binding
protein48,49 that stabilizes TfR mRNA and
represses ferritin synthesis in cells50,51. The
iron-sulfur cluster of IRP1 is readily destabi52,53, including nitric oxide54
lized by oxidants
and hydrogen peroxide55, and cluster degradaloop
tion produces apoprotein that binds IREs and
lacks iron and sulfur (Fig. 4a)44,56.
Numerous cross-linking, mutagenesis and
chemical modification studies have indicated
that the IRE binding site of IRP1 overlaps
with the aconitase active site48,49,57. Given the
known dimensions of several RNA stem-loop
structures, it was hypothesized that IRE bindLower stem
ing could occur within the active site cleft of
IRP1 if the fourth domain of the protein, which
is connected to domains one through three by
a flexible hinge linker, were to swing open. Figure 2 IRE secondary structure. (a) A schematic of a consensus IRE is shown. The IRE contains a
Further mutagenesis58 and footprinting of the six-residue loop, usually with the sequence CAGYGX, where Y represents U or C and X represents any
IRE-IRP interactions59 indicated that residues residue except G. The upper and lower stems are composed of base pairs (bp) of variable sequence
near the entrance to the active site cleft on both (N-N′) that are separated by an unpaired C. (b) In the NMR solution structure of a consensus IRE, a bp
sides are important in IRE binding. Figure 4b forms between C1 and G5, and A2 stacks on G5 in the conserved loop sequence CAGUGX (modified
from ref. 11, courtesy of K. Addess, with permission). The helical upper and lower stems have an
shows the structure of IRP1 with an intact iron- A-form conformation, and both the bulge C and the unpaired G residue at position 3 in the loop are
46 as well as another view of the
disordered in solution. The 5-bp upper stem most likely functions as a molecular ruler that orients and
protein in which domain 4 has been displaced correctly distances the bulge C from residues in the loop, allowing flexible residues to participate in
to accommodate docking of the IRE (created sequence-specific interactions between the IRE and IRPs. 408 VOLUME 2 NUMBER 8 AUGUST 2006 NATURE CHEMICAL BIOLOGY © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology PERSPECTIVE
bind IREs, and the apoprotein form, which lacks an iron-sulfur cluster
and binds IREs. It is now well accepted that the ‘iron-sulfur cluster switch’
is an important determinant of whether IRP1 has cytosolic aconitase or
IRE-binding activity1,6,7,53,61. Synthesis of the iron-sulfur cluster requires
iron, sulfur and dedicated biosynthetic proteins, whereas cluster turnover
results mainly from cluster oxidation and spontaneous degradation. The
strength and capacity of the forces that enhance new synthesis relative
to that of the forces that favor cluster degradation determine the way in
which IRP1 functions (Fig. 4a). Thus, to understand IRP1 function, it is
important to understand the factors involved in assembly and turnover
of iron-sulfur clusters in mammalian cells.
Mammalian iron-sulfur cluster assembly
Assembly of iron-sulfur clusters is a complex process involving many
enzymes and scaffold proteins62,63, and mammalian iron-sulfur clusters
are synthesized by homologs of bacterial and yeast proteins64 (Fig. 5).
Through alternative splicing and alternative initiation, mammalian cells
generate cytosolic isoforms of iron-sulfur cluster assembly proteins that
directly facilitate assembly of the iron-sulfur cluster of IRP1 in the cytosol65,66. Mammalian iron-sulfur assembly proteins known to be present
in both mitochondria and cytosol include the cysteine desulfurase that
provides sulfur in the correct oxidation state67, the primary scaffold on
which nascent clusters are assembled (iron sulfur cluster U assembly protein)68 and the scaffold protein NFU (a protein required for maturation
of FeS clusters of nitrogenase in nitrogen-fixing bacteria)69. Other proteins involved in iron-sulfur cluster assembly include (i) frataxin, which
may serve as an iron donor, (ii) reducing proteins such as ferredoxin
and glutaredoxin and (iii) chaperone and co-chaperone proteins, which
facilitate protein folding reactions when nascent clusters are transferred
from scaffold to recipient proteins (Fig. 5). Clearly, iron-sulfur cluster
assembly requires sufficient sources of sulfur and iron, and deficiency
of iron-sulfur cluster assembly proteins, cysteine or iron can potentially
inhibit iron-sulfur cluster assembly. Expression of the iron-sulfur cluster
assembly scaffold protein ISCU decreases in iron-deficient cells65, thereby
offering the possibility that the iron-sulfur switch of IRP1 may be regulated not only through iron availability, but also through regulation of
the expression of iron-sulfur cluster assembly proteins.
There are several recent examples in which impairment of the ironsulfur cluster assembly machinery activates the IRE binding activity
of IRP1 in a physiologically relevant setting. In zebrafish, mutations
in glutaredoxin 5 (an enzyme important in iron-sulfur cluster biogenesis)70,71 cause heme deficiency, profound anemia and early death.
Rescue experiments indicate that the first step of heme biosynthesis
is repressed by binding of IRP1 to the eALAS transcript of zebrafish,
which, as in mammals, contains an IRE in its 5′ UTR. The zebrafish
studies establish an important genetic linkage between heme biosynthesis and the iron-sulfur cluster biogenesis of IRP1 (ref. 72), and they
constitute an important independent confirmation of the iron-sulfur
switch model of IRP1 regulation73. In mammalian cells, knockdown
of ISCU markedly reduces mitochondrial aconitase activity and shifts
IRP1 from the aconitase form to the IRE-binding form. Moreover, specific knockdown of the cytosolic ISCU isoform shows that the ironsulfur switch of IRP1 can be activated by interfering directly with the
cytosolic iron-sulfur cluster assembly machinery65. However, it is not
yet clear how many iron-sulfur assembly proteins are present in mammalian cytosol.
Iron-sulfur cluster disassembly
The state of the iron-sulfur switch of IRP1 depends not only on synthesis of the iron-sulfur cluster but also on the rate of cluster turnover.
The iron-sulfur cluster of IRP1 can be oxidized and destabilized by NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 8 AUGUST 2006 oxygen, nitric oxide and peroxynitrite74, and it can be indirectly disassembled by hydrogen peroxide–mediated activation of a signaling
pathway75. In macrophages in which IRP1 could be exposed to endogenously generated reactive oxygen species and nitric oxide, there is little
apparent cytosolic aconitase activity76. Notably, silencing of cytosolic
superoxide dismutase in Drosophila melanogaster results in activation of
cytosolic aconitase but not mitochondrial aconitase, whereas silencing
of mitochondrial superoxide dismutase (SOD) decreases mitochondrial
but not cytosolic aconitase activity77, implying that superoxide can
be an important cause of intracellular iron-sulfur cluster disassembly.
Similarly, in mice with cytosolic SOD deficiency, cytosolic aconitase
activity decreases as IRE binding activity concomitantly increases78. In
yeast with cytosolic SOD deficiency, iron-sulfur enzymes in both the
cytosol and the mitochondria have decreased activity79. Thus, results
in fly, yeast and mouse model systems implicate superoxide as a potentially potent cause for disassembly of the iron-sulfur clusters of both
mitochondrial and cytosolic aconitases. Verona ( +41)
Paris 1 ( +41)
London 1 ( +41)
London 2 ( +36) Paris 2 ( +32)
Pavia 1 ( +32) Pavia 2
(+18 and +22) (+1)
Figure 3 Mutations of the ferritin L-chain IRE cause HHCS. Multiple
mutations in the ferritin L-chain transcript interfere with IRP binding,
resulting in derepression of ferritin L-chain translation, high serum ferritin
concentrations and cataract formation. The ferritin L-chain transcriptional
start site (+1) is shown, and the first 77 nucleotides are depicted in an
extended stem-loop structure with residues numbered in sequence14.
Multiple mutations are labeled to show the nucleotide change, along
with the name of the city in which patients affected by the mutation
were first identified. Mutations affect the loop and the upper and lower
stems, including a G32U substitution in the Paris 2 mutation and a G32A
substitution in the Pavia 1 mutation (shown), which is also the site of a
recently described G32C mutation21 that affects the potential base pair
formation of G32 within a region of the ferritin IRE that is often represented
as an unpaired residue within a three-nucleotide ‘bulge’14. 409 PERSPECTIVE
Bulged C Fe2+ released © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology O2 or other
assemby enzymes G b
(2–240) Domain 1
(2–240) Domain 2
(241–368) 2 Domain 2
(241–368) 2 Linker
(369–592) 889 Domain 4
(655–889) Domain 3
(369–592) IRE [4Fe-4S] Figure 4 The iron-sulfur switch of IRP1. (a) A
cartoon showing the iron-sulfur switch of IRP1.
IRP1 is a bifunctional protein that can exist as
a functional cytosolic aconitase, interconverting
citrate and isocitrate, or as an apoprotein that
binds IREs. Studies indicate that the active site
cleft responsible for aconitase activity overlaps
extensively with the region that binds to IREs.
Thus, IRP1 is bifunctional, and the ratio of the
holoprotein and apoprotein forms most likely
determines the way IRP1 functions in cells.
Instability of the iron-sulfur prosthetic group
may be a key determinant of the ratio between
holoprotein and apoprotein. Numerous enzymes
and proteins facilitate iron-sulfur cluster assembly
and formation of active cytosolic aconitase.
(b) Left, the IRP1 structure with its cluster; right,
IRP1 modeled with the IRE bound. Structures
are depicted according to the recently solved
holoprotein structure46; illustrations created by
J. Dupuy. Domains are labeled; domains 1–3
are connected to domain 4 by a flexible hinge
linker (black) that is depicted as seen in the
crystal structure (left) or in a conformation that
could accommodate the IRE, based on structural
modeling (right) in which the position of domain
4 is moved to accommodate the IRE within an
enlarged cleft. Domain 4
(655–889) Some IRP1s may lack function as either a cytosolic aconitase or an IRE
binding protein80, but the physiologic relevance and size of this functionless pool of IRP1 is not yet clear. Nitration of IRP1 in activated macrophages leads to inactivation of the aconitase and IRE binding activities
of IRP1 without causing degradation81. In addition, iron-dependent
degradation of IRP1 occurs in some cells and can be enhanced by phosphorylation at Ser138 (ref. 78), thereby providing a potential link between
growth signaling pathways and IRP1 activity. Phosphorylation of IRP1
at Ser711 can also modulate aconitase activity in vivo82,83. Thus, various
factors that do not directly affect iron-sulfur cluster assembly and disassembly may modify operation of the iron-sulfur switch.
The role of IRP1 in mammalian physiology
Because IRP1 is abundant in animal tissues and regulates its IRE binding activity in tissue culture and in rats subjected to iron depletion84,
investigators have assumed that IRP1 is important in intracellular iron
regulation. However, genetic ablation studies have revealed that the
homologous protein, IRP2, is important in the regulation of iron metabolism in mice85,86, whereas ablation of IRP1 has little effect on regulation
of tissue iron homeostasis, except in kidney and brown fat, in which IRP1
expression is very high87. Unlike in cells grown in room air, most IRP1
in animal tissues is in the aconitase form, and IRP1 does not convert to
the IRE-binding form in cells given a low-iron diet that is sufficient to
activate IRP2 (ref. 87).Even when IRP2 is absent, IRP1 binding activity
does not increase, whereas when IRP1 is absent, IRP2 binding activity
and concentration increases in a compensatory fashion87,88.
Thus, it seems that the primary physiologic role of IRP1 may be to
function as a cytosolic aconitase. By interconverting citrate and isocitrate
in the cytosol, cytosolic aconitase may allow cells to balance the amount
of reduced nicotinamide adenine dinucleotide phosphate (NADPH) generated by cytosolic isocitrate dehydrogenase with the amount of acetyl
coenzyme A generated from cytosolic citrate by the citrate lyase reaction.
Fatty acid synthesis requires (i) the acetyl coenzyme A building blocks 410 derived from citrate and (ii) NADPH, which is generated by cytosolic
isocitrate dehydrogenase when it catalyzes conversion of isocitrate to
2-oxoglutarate. Recent studies indicate that cytosolic isocitrate dehydrogenase is one of the main sources of NADPH in mammalian cells89. In
energy-rich cells, large NADH to NAD+ ratios repress mitochondrial
isocitrate dehydrogenase activity90, leading to accumulation of citrate
and isocitrate in the mitochondrial matrix and driving export of these
citric acid intermediates to the cytosol through the tricarboxylic acid
transporter, where they can serve as fatty acid precursors91. Notably,
IRP1 is most highly expressed in brown fat, liver, kidney and testicular
epididymal cells, potentially important sites for energy storage and fatty
The difference between the potential of IRP1 to regulate iron metabolism and its actual role in animals is most likely attributable to the role of
oxygen in destabilizing iron-sulfur clusters. In tissue culture, cells are usually exposed to the high atmospheric oxygen concentrations of room air,
whereas in animals, oxygen concentrations are much lower, in the range
of 3–6%88. IRP1 provides most of the IRE binding activity detected in
cells grown in room air, whereas IRP1 converts to the cytosolic aconitase
form at the low oxygen tensions that prevail in mammalian tissues and is
less important than IRP2 in regulation of iron metabolism (Fig. 6). Thus,
turnover of the labile iron-sulfur cluster is likely to be markedly lower in
animal tissues than in tissue culture cells, and cluster turnover in animal
tissues may depend more on the reactive oxygen species and nitric oxide
produced by stimuli such as infection and inflammation53.
Role of IRP2 in regulation of iron metabolism
IRP2 is a ubiquitously expressed regulatory protein that has a central role
in mammalian iron metabolism85,86. Mice that lack IRP2 develop anemia
due to insufficient erythroid expression of TfR, which combines with
overexpression of ferritin to deplete cells of iron that is needed for heme
synthesis. IRP2−/− mice do not appropriately repress translation of eALAS,
which has an IRE at its 5′ end; consequently, they produce 200-fold more VOLUME 2 NUMBER 8 AUGUST 2006 NATURE CHEMICAL BIOLOGY PERSPECTIVE
of the porphyrin heme precursor protoporphyISCS
rin IX than do wild-type mice25. In adulthood,
−/− mice develop progressive neurodegenIRP2
erative disease associated with degeneration of
axons followed by death of neuronal cell bodies
S from cysteine
in cells that overexpress ferritin and synthesize
too little TfR85,92,93. Anemia without progressive neurodegeneration has been reported in
one IRP2−/− mouse model86, but neurodegenTarget protein
eration is most likely the primary consequence
of IRP loss, given that both neurodegeneration
and anemia are exacerbated in IRP1+/− IRP2−/−
mice compared with IRP2−/− mice (Fig. 7a–f)92,
a result indicating that IRP1 and IRP2 function
in the same pathway and that there is a gene
Although IRP1 and IRP2 are equally able to
regulate ferritin translation in vitro94 and are Figure 5 Proposed schematic of mammalian iron-sulfur cluster assembly. The cysteine desulfurase
both ubiquitously expressed, IRP2 dominates ISCS generates sulfur, which it donates to ISCU. ISCU binds iron, perhaps donated by frataxin, and
regulation of iron homeostasis87. The reason serves as a scaffold on which nascent iron-sulfur clusters are assembled. ISCA and NFU may also
for this domination is likely to be intimately function as scaffold proteins. Ferredoxin (FDX) and glutaredoxin 5 (GRX) provide reducing equivalents,
related to IRP2’s unique mechanism for sens- and the chaperones HSCA and HSCB (heat shock cognate proteins A and B, respectively) are likely to
ing and regulating iron concentrations. In iron- enhance scaffold folding and transfer of clusters to recipient apoproteins such as IRP1 (refs. 63,64).
replete cells, IRP2 is efficiently degraded in an
iron-dependent manner, and its concentration
is not regulated by an iron-sulfur switch17,41,42. In addition, cells acti- is also possible, but unlikely, that IRP2 transiently assembles an ironvated by expression of the c-myc oncogene increase expression of IRP2 sulfur cluster17.
mRNA, indicating that regulation of IRP2 expression may be affected by
A critical feature that allows IRP2 to predominate in the regulation of
the growth status of cells95. Thus, regulation of transcription and protein iron metabolism is IRP2’s relative stability and IRE-binding activity at the
low oxygen concentrations that prevail in mammalian tissues. IRP1, in
turnover most likely affect IRP2 expression.
contrast, is mainly active as an aconitase at these low oxygen concentrations (Fig. 6). Another important difference is that IRP2 can compensate
Mechanism of iron-dependent degradation of IRP2
The mechanism by which IRP2 undergoes iron-dependent degrada- for loss of IRP1 (ref. 88), perhaps because loss of IRP1 results in mild iron
tion is incompletely understood. Relative to IRP1, IRP2 contains an deficiency due to minor decreases in TfR expression and increases in ferextra cysteine-rich exon. Previously referred to as the iron-dependent ritin, and decreased cytosolic iron concentrations partially stabilize IRP2.
degradation domain, this exon has been thought to be responsible for Though recent data on regulation of ISCU indicate that iron deficiency
characteristic iron-dependent degradation owing to its ability to facili- decreases cellular ISCU expression and may thereby reduce the amount
tate iron-dependent oxidation96, ubiquitination and proteasomal deg- of IRP1 that has an iron-sulfur cluster65, regulation of iron-sulfur cluster
radation97,98. However, the initial steps in iron-dependent degradation assembly proteins has yet to be characterized in intact animals.
of IRP2 are not well characterized, and the role of the iron-dependent
degradation domain has been questioned99,100. Other molecules or fac- The physiologic importance of the IRE-IRP regulatory system
tors that may signal the iron-replete state and facilitate degradation of Developing embryos that lack both copies of IRP1 and IRP2 die at the
IRP2 include heme100–103, 2-oxoglutarate–dependent oxygenases99,104 blastocyst stage, before implantation106. This embryonic lethality of the
and phosphorylation status105, and the contribution of various pathways IRP1−/− IRP2−/− genotype underscores the fact that the IRP-IRE regulato IRP2 degradation may depend in part on the cells involved103. It is tory system is critical for regulation of iron metabolism and that IRP1
possible that IRP2 has evolved to ‘sense’ cytosolic iron concentrations and IRP2 have redundant functions. Their redundancy is further underthrough multiple pathways: direct binding of iron or heme, as well as scored by the fact that the neurodegeneration and anemia of IRP2−/−
activation of cytosolic oxygenases, may combine to robustly target IRP2 mice is greatly exacerbated in animals that also lack at least one copy
for ubiquitination and proteasomal degradation in iron-replete cells. It of IRP1 (Fig. 7)25,92. The gene dosage effect of IRP loss is illustrated by
– + Figure 6 IRP2 has greater IRE-binding activity than IRP1 at the
characteristically low oxygen concentrations found in mammalian tissues.
At these low oxygen concentrations IRP2 is highly active as an IRE-binding
protein, whereas IRP1 acts mainly as a cytosolic aconitase and has low IRE
binding activity. The schematic shows IRP binding activity over a range
of oxygen concentrations, along with markings that indicate the reported
mammalian tissue oxygen concentrations of bone marrow, spleen and brain
(yellow arrows). The oxygen concentrations of many other tissues are between
3% and 6% (the area between the two vertical black arrows on the x axis),
whereas most cells grown in traditional tissue culture flasks are exposed to
oxygen concentrations closer to the 21% oxygen concentration of room air. NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 8 AUGUST 2006 IRE binding activity © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology 3 IRP1-IRE binding activity 1%
Brain Oxygen concentration 21%
Tissue culture 411 PERSPECTIVE Wild type IRP2–/– IRP2–/– IRP1+/– a b c d e f © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology Ferric staining Axonal
degeneration 2 mm
IRP2–/– Wild type g h Toluidine blue 10 µM Figure 7 Neurodegeneration in adult IRP2−/− and IRP1+/− IRP2−/− animals is characterized by ferric iron accumulations in distinctive white-matter areas of
the brain and by axonal degeneration in the affected white-matter areas. (a–f) Coronal sections of whole mouse brains through the cerebellum show little
ferric iron accumulation or axonal degeneration in wild-type mice (a,d). However, ferric iron accumulations (golden-brown stain in a–c) are present in IRP2−/−
mice (b) and are markedly increased in IRP2−/− IRP1+/− mice (c), and there is concomitant axonal degeneration in IRP2−/− mice (e), which is more marked in
IRP2−/− IRP1+/− mice (f), as indicated by deposition of black thread-like silver deposits within white matter in degenerating axons. A green counterstain for
nuclei heavily stains regions that are rich in cell bodies (traditional gray matter) in a–c, whereas a red nuclear counterstain is used in d–f to allow visualization
of distinctive landmarks near the back of the brain, particularly the cerebellar folia. (g,h) In brain tissue fixed in epoxy and stained with toluidine blue for
improved morphologic visualization at high magnification, myelin-dense bodies indicative of axonal degeneration are commonly found (arrow) in IRP2−/− (h)
but not in wild-type mice (g). In this stain, normal axons appear as blue rings with white centers; the thick blue rim represents the myelin sheath, whereas
the white center represents the normal axon. Myelin-dense bodies are created when axons degenerate and the myelin sheath collapses to fill the space
formerly occupied by an axon. Figures reproduced from ref. 92 with permission from Blackwell Publishing. ferric iron accumulations, which are detectable in many axon-rich regions
of the brain in 1-year-old IRP2−/− mice (Fig. 7c) and are pronounced in
age-matched IRP1+/− IRP2−/− mice (Fig. 7e) but are not detectable in agematched wild-type mice (Fig. 7a). The ferric iron is most likely sequestered by ferritin and is relatively inaccessible, even though increased ferric
iron staining is usually thought to signify increased iron availability85,92.
In cells, the combination of ferritin overexpression with decreased
TfR1 expression may result in a state of functional iron deficiency that
adversely affects axons, which depend on iron-replete mitochondria to
generate the ATP that supports axonal transport and function. Notably,
axons in white matter areas of the brain that accumulate ferric iron show
degeneration, as evidenced by a special staining procedure known as the
amino cupric silver stain, in which black deposits of silver are observed
in IRP2−/− and IRP1+/− IRP2−/− brains (Fig. 7e,f)92. Intact axons of the 412 wild type prevent penetration of silver (Fig. 7b), whereas degenerating
axons allow the silver to penetrate and bind to negatively charged neurofilaments, creating threadlike black deposits that follow the course of
axons. Ultimately, complete loss of axonal integrity results in collapse of
the myelin sheath around axonal remnants in mutant mice (Fig. 7h), but
not in wild-type mice (Fig. 7g).
Notably, animals having some residual IRP activity (IRP1+/− IRP2−/−
animals) can survive to adulthood with apparent functional compromise
of only the hematopoietic and neurologic systems, whereas other cells
and tissues are spared. In neurons, swelling and inflammation initially
affects axons, but ultimately neuronal cell bodies die, particularly in the
substantia nigra, the area of the brain affected in Parkinson disease92. The
cause of neuronal dysfunction and death is not yet clear, but based on an
analogy to erythroid cells, it is possible that iron deficiency may adversely VOLUME 2 NUMBER 8 AUGUST 2006 NATURE CHEMICAL BIOLOGY PERSPECTIVE © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology affect neurons by compromising synthesis of the iron-sulfur complexes
of the mitochondrial respiratory chain.
The fact that IRP2 loss causes adult-onset neurodegenerative disease
with Parkinsonian features raises the possibility that IRP2 dysfunction
will prove to be a cause of adult-onset human neurodegenerative disease. Affected individuals carrying IRP2 mutations are also likely to have
microcytic anemia and elevated red cell protoporphyrin IX concentrations, although the movement disorder and anemia can be very subtle if
only one IRP2 allele is inactivated85.
Animal studies indicate that the IRP-IRE regulatory system is important
in physiology. Future studies are likely to bring more knowledge about
mammalian iron-sulfur cluster assembly and the factors that operate
the iron-sulfur cluster switch of IRP1. Much remains to be determined
about the mechanisms by which IRP2 is targeted for iron-dependent
degradation and those by which IRP transcription is affected by growth
signals. The neurodegeneration of IRP2−/− mice provides a useful model
in which the causes of neuronal death can be dissected. If functional
iron deficiency causes mitochondrial dysfunction, which in turn causes
free radical stress and failure of energy production in axons, this central insight may provide understanding of the pathophysiology of many
neurodegenerative diseases in which iron misregulation is suspected to
have a role.
I thank J. Dupuy for drawing the figure of IRP1, modeling the IRP1-IRE interaction
and generously sharing his insights about IRP1 structure, W.H. Tong for creating
the iron-sulfur cluster biogenesis schematic and my scientific colleagues for their
excellent work and help. This work was supported by the intramural program of the
National Institute of Child Health and Human Development.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
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17. 18. 19. 20. 21. 22.
25. 26. 27. 28. 29. 30.
11. 12. 13. 14. Hentze, M.W., Muckenthaler, M.U. & Andrews, N.C. Balancing acts: molecular
control of mammalian iron metabolism. Cell 117, 285–297 (2004).
Ganz, T. Hepcidin in iron metabolism. Curr. Opin. Hematol. 11, 251–254
Aisen, P. Transferrin, the transferrin receptor, and the uptake of iron by cells. Met.
Ions Biol. Syst. 35, 585–631 (1998).
Aisen, P. Transferrin receptor 1. Int. J. Biochem. Cell Biol. 36, 2137–2143
Harrison, P.M. & Arosio, P. The ferritins: molecular properties, iron storage function
and cellular regulation. Biochim. Biophys. Acta 1275, 161–203 (1996).
Rouault, T. & Klausner, R. Regulation of iron metabolism in eukaryotes. Curr. Top.
Cell. Regul. 35, 1–19 (1997).
Pantopoulos, K. Iron metabolism and the IRE/IRP regulatory system: an update.
Ann. NY Acad. Sci. 1012, 1–13 (2004).
Klausner, R.D., Rouault, T.A. & Harford, J.B. Regulating the fate of mRNA: the
control of cellular iron metabolism. Cell 72, 19–28 (1993).
Gruer, M.J., Artymiuk, P.J. & Guest, J.R. The aconitase family: three structural
variations on a common theme. Trends Biochem. Sci. 22, 3–6 (1997).
Beinert, H., Kennedy, M.C. & Stout, D.C. Aconitase as iron-sulfur protein, enzyme,
and iron-regulatory protein. Chem. Rev. 96, 2335–2373 (1996).
Addess, K.J., Basilion, J.P., Klausner, R.D., Rouault, T.A. & Pardi, A.J. Structure
and dynamics of the iron responsive element RNA: implications for binding of the
RNA by iron regulatory proteins. J. Mol. Biol. 274, 72–83 (1997).
Gdaniec, Z., Sierzputowska-Gracz, H. & Theil, E.C. Iron regulatory element and
internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine
binding, molecular modeling, and NMR spectroscopy. Biochemistry 37, 1505–1512
Dix, D.J., Lin, P.N., McKenzie, A.R., Walden, W.E. & Theil, E.C. The influence of
the base-paired flanking region on structure and function of the ferritin mRNA iron
regulatory element. J. Mol. Biol. 231, 230–240 (1993).
Allerson, C.R., Cazzola, M. & Rouault, T.A. Clinical severity and thermodynamic
effects of iron-responsive element mutations in hereditary hyperferritinemia-cataract
syndrome. J. Biol. Chem. 274, 26439–26447 (1999). NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 8 AUGUST 2006 34. 35. 36.
37. 38. 39. 40.
41. 42. 43.
44. Henderson, B.R., Menotti, E. & Kuhn, L.C. Iron regulatory proteins 1 and 2 bind
distinct sets of RNA target sequences. J. Biol. Chem. 271, 4900–4908 (1996).
Butt, J. et al. Differences in the RNA binding sites of iron regulatory proteins and
potential target diversity. Proc. Natl. Acad. Sci. USA 93, 4345–4349 (1996).
Meehan, H.A. & Connell, G.J. The hairpin loop but not the bulged C of the iron
responsive element is essential for high affinity binding to iron regulatory protein-1.
J. Biol. Chem. 276, 14791–14796 (2001).
Menotti, E., Henderson, B.R. & Kuhn, L.C. Translational regulation of mRNAs with
distinct IRE sequences by iron regulatory proteins 1 and 2. J. Biol. Chem. 273,
Ke, Y., Wu, J., Leibold, E.A., Walden, W.E. & Theil, E.C. Loops and bulge/loops in
iron-responsive element isoforms influence iron regulatory protein binding. Finetuning of mRNA regulation. J. Biol. Chem. 273, 23637–23640 (1998).
Theil, E.C. & Eisenstein, R.S. Combinatorial mRNA regulation: iron regulatory
proteins and iso-iron-responsive elements (iso-IREs). J. Biol. Chem. 275, 40659–
Ismail, A.R., Lachlan, K.L., Mumford, A.D., Temple, I.K. & Hodgkins, P.R. Hereditary
hyperferritinemia cataract syndrome: ocular, genetic, and biochemical findings. Eur.
J. Ophthalmol. 16, 153–160 (2006).
Hentze, M.W. et al. Identification of the iron-responsive element for the translational
regulation of human ferritin mRNA. Science 238, 1570–1573 (1987).
Leibold, E.A. & Munro, H.N. Cytoplasmic protein binds in vitro to a highly conserved
sequence in the 5′ untranslated region of ferritin heavy- and light-subunit mRNAs.
Proc. Natl. Acad. Sci. USA 85, 2171–2175 (1988).
Melefors, O. et al. Translational control of 5-aminolevulinate synthase mRNA by ironresponsive elements in erythroid cells. J. Biol. Chem. 268, 5974–5978 (1993).
Cooperman, S.S. et al. Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood
106, 1084–1091 (2005).
Kim, H.Y., LaVaute, T., Iwai, K., Klausner, R.D. & Rouault, T.A. Identification of
a conserved and functional iron-responsive element in the 5′UTR of mammalian
mitochondrial aconitase. J. Biol. Chem. 271, 24226–24230 (1996).
Gray, N.K., Pantopoulos, K., Dandekar, T., Ackrell, B.A. & Hentze, M.W. Translational
regulation of mammalian and Drosophila citric-acid cycle enzymes via iron-responsive elements. Proc. Natl. Acad. Sci. USA 93, 4925–4930 (1996).
Schalinske, K.L., Chen, O.S. & Eisenstein, R.S. Iron differentially stimulates translation of mitochondrial aconitase and ferritin mRNAs in mammalian cells. Implications
for iron regulatory proteins as regulators of mitochondrial citrate utilization. J. Biol.
Chem. 273, 3740–3746 (1998).
Kohler, S.A., Henderson, B.R. & Kuhn, L.C. Succinate dehydrogenase b mRNA of
Drosophila melanogaster has a functional iron-responsive element in its 5′-untranslated region. J. Biol. Chem. 270, 30781–30786 (1995).
Abboud, S. & Haile, D.J. A novel mammalian iron-regulated protein involved in
intracellular iron metabolism. J. Biol. Chem. 275, 19906–19912 (2000).
Gunshin, H. et al. Cloning and characterization of a mammalian proton-coupled
metal-ion transporter. Nature 388, 482–488 (1997).
Kohler, S.A., Menotti, E. & Kuhn, L.C. Molecular cloning of mouse glycolate oxidase.
High evolutionary conservation and presence of an iron-responsive element-like
sequence in the mRNA. J. Biol. Chem. 274, 2401–2407 (1999).
Gunshin, H. et al. Iron-dependent regulation of the divalent metal ion transporter.
FEBS Lett. 509, 309–316 (2001).
Recalcati, S., Tacchini, L., Alberghini, A., Conte, D. & Cairo, G. Oxidative stressmediated down-regulation of rat hydroxyacid oxidase 1, a liver-specific peroxisomal
enzyme. Hepatology 38, 1159–1166 (2003).
Beaumont, C. et al. Mutation in the iron responsive element of the L ferritin mRNA
in a family with dominant hyperferritinaemia and cataract. Nat. Genet. 11, 444–446
Cazzola, M. & Skoda, R.C. Translational pathophysiology: a novel molecular mechanism of human disease. Blood 95, 3280–3288 (2000).
Rouault, T.A. et al. Cloning of the cDNA encoding an RNA regulatory protein–the
human iron-responsive element-binding protein. Proc. Natl. Acad. Sci. USA 87,
Patino, M.M. & Walden, W.E. Cloning of a functional cDNA for the rabbit ferritin
mRNA repressor protein: demonstration of a tissue specific pattern of expression.
J. Biol. Chem. 267, 19011–19016 (1992).
Yu, Y., Radisky, E. & Leibold, E.A. The iron-responsive element binding protein:
purification, cloning and regulation in rat liver. J. Biol. Chem. 267, 19005–19010
Hirling, H. et al. Expression of active iron regulatory factor from a full-length human
cDNA by in vitro transcription/translation. Nucleic Acids Res. 20, 33–39 (1992).
Guo, B., Yu, Y. & Leibold, E.A. Iron regulates cytoplasmic levels of a novel ironresponsive element-binding protein without aconitase activity. J. Biol. Chem. 269,
Samaniego, F., Chin, J., Iwai, K., Rouault, T.A. & Klausner, R.D. Molecular characterization of a second iron responsive element binding protein, iron regulatory protein
2 (IRP2): structure, function and post-translational regulation. J. Biol. Chem. 269,
Kaptain, S. et al. A regulated RNA binding protein also possesses aconitase activity.
Proc. Natl. Acad. Sci. USA 88, 10109–10113 (1991).
Kennedy, M.C., Mende-Mueller, L., Blondin, G.A. & Beinert, H. Purification and
characterization of cytosolic aconitase from beef liver and its relationship to the
iron-responsive element binding protein (IRE-BP). Proc. Natl. Acad. Sci. USA 89,
11730–11734 (1992). 413 PERSPECTIVE
48. © 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology 49. 50. 51.
55. 56. 57. 58.
65. 66. 67. 68.
69. 70. 71. 72.
74. 75. 414 Zheng, L., Andrews, P.C., Hermodson, M.A., Dixon, J.E. & Zalkin, H. Cloning and
structural characterization of porcine heart aconitase. J. Biol. Chem. 265, 2814–
Dupuy, J. et al. Crystal structure of human iron regulatory protein 1 as cytosolic
aconitase. Structure 14, 129–139 (2006).
Robbins, A.H. & Stout, C.D. The structure of aconitase. Proteins 5, 289–312
Hirling, H., Henderson, B.R. & Kuhn, L.C. Mutational analysis of the [4Fe-4S]-cluster
converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.
EMBO J. 13, 453–461 (1994).
Philpott, C.C., Klausner, R.D. & Rouault, T.A. The bifunctional iron-responsive element
binding protein/cytosolic aconitase: the role of active-site residues in ligand binding
and regulation. Proc. Natl. Acad. Sci. USA 91, 7321–7325 (1994).
DeRusso, P.A. et al. Expression of a constitutive mutant of iron regulatory protein 1
abolishes iron homeostasis in mammalian cells. J. Biol. Chem. 270, 15451–15454
Wang, J. & Pantopoulos, K. Conditional derepression of ferritin synthesis in cells
expressing a constitutive IRP1 mutant. Mol. Cell. Biol. 22, 4638–4651 (2002).
Rouault, T.A. & Klausner, R.D. Iron-sulfur clusters as biosensors of oxidants and iron.
Trends Biochem. Sci. 21, 174–177 (1996).
Cairo, G., Recalcati, S., Pietrangelo, A. & Minotti, G. The iron regulatory proteins:
targets and modulators of free radical reactions and oxidative damage. Free Radic.
Biol. Med. 32, 1237–1243 (2002).
Bouton, C. & Drapier, J.C. Iron regulatory proteins as NO signal transducers. Sci. STKE
2003, pe17 (2003).
Caltagirone, A., Weiss, G. & Pantopoulos, K. Modulation of cellular iron metabolism by
hydrogen peroxide. Effects of H2O2 on the expression and function of iron-responsive
element-containing mRNAs in B6 fibroblasts. J. Biol. Chem. 276, 19738–19745
Haile, D.J. et al. Cellular regulation of the iron-responsive element binding protein:
disassembly of the cubane iron-sulfur cluster results in high affinity RNA binding.
Proc. Natl. Acad. Sci. USA 89, 11735–11739 (1992).
Basilion, J.P., Rouault, T.A., Massinople, C.M., Klausner, R.D. & Burgess, W.H. The
iron-responsive element-binding protein: localization of the RNA binding site to the
aconitase active-site cleft. Proc. Natl. Acad. Sci. USA 91, 574–578 (1994).
Kaldy, P., Menotti, E., Moret, R. & Kuhn, L.C. Identification of RNA-binding surfaces
in iron regulatory protein-1. EMBO J. 18, 6073–6083 (1999).
Gegout, V. et al. Ligand-induced structural alterations in human iron regulatory protein1 revealed by protein footprinting. J. Biol. Chem. 274, 15052–15058 (1999).
Selezneva, A.I., Cavigiolio, G., Theil, E.C., Walden, W.E. & Volz, K. Crystallization
and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex
with ferritin IRE RNA. Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 62,
Eisenstein, R.S. Iron regulatory proteins and the molecular control of mammalian iron
metabolism. Annu. Rev. Nutr. 20, 627–662 (2000).
Johnson, D.C., Dean, D.R., Smith, A.D. & Johnson, M.K. Structure, function, and
formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281
Lill, R. & Muhlenhoff, U. Iron-sulfur-protein biogenesis in eukaryotes. Trends Biochem.
Sci. 30, 133–141 (2005).
Rouault, T.A. & Tong, W.H. Opinion: iron-sulphur cluster biogenesis and mitochondrial
iron homeostasis. Nat. Rev. Mol. Cell Biol. 6, 345–351 (2005).
Tong, W.H. & Rouault, T.A. Functions of mitochondrial ISCU and cytosolic ISCU
in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab. 3,
Li, K., Tong, W.H., Hughes, R.M. & Rouault, T.A. Roles of the mammalian cytosolic
cysteine desulfurase, ISCS, and scaffold protein, ISCU, in iron-sulfur cluster assembly.
J. Biol. Chem. 281, 12344–12351 (2006).
Land, T. & Rouault, T.A. Targeting of a human iron-sulfur cluster assembly enzyme,
nifs, to different subcellular compartments is regulated through alternative AUG utilization. Mol. Cell 2, 807–815 (1998).
Tong, W.H. & Rouault, T. Distinct iron-sulfur cluster assembly complexes exist in the
cytosol and mitochondria of human cells. EMBO J. 19, 5692–5700 (2000).
Tong, W.H., Jameson, G.N., Huynh, B.H. & Rouault, T.A. Subcellular compartmentalization of human Nfu, an iron-sulfur cluster scaffold protein, and its ability to assemble
a [4Fe-4S] cluster. Proc. Natl. Acad. Sci. USA 100, 9762–9767 (2003).
Rodriguez-Manzaneque, M.T., Tamarit, J., Belli, G., Ros, J. & Herrero, E. Grx5 is a
mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes. Mol. Biol.
Cell 13, 1109–1121 (2002).
Molina-Navarro, M.M., Casas, C., Piedrafita, L., Belli, G. & Herrero, E. Prokaryotic
and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in
the biogenesis of Fe/S clusters in yeast mitochondria. FEBS Lett. 580, 2273–2280
Wingert, R.A. et al. Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for
vertebrate haem synthesis. Nature 436, 1035–1039 (2005).
Rouault, T.A. Linking physiological functions of iron. Nat. Chem. Biol. 1, 193–194
Cairo, G., Ronchi, R., Recalcati, S., Campanella, A. & Minotti, G. Nitric oxide and
peroxynitrite activate the iron regulatory protein-1 of. Biochemistry 41, 7435–7442
Mueller, S., Pantopoulos, K., Hubner, C.A., Stremmel, W. & Hentze, M.W. IRP1 activation by extracellular oxidative stress in the perfused rat liver. J. Biol. Chem. 276,
23192–23196 (2001). 76. 77.
79. 80. 81. 82. 83. 84. 85. 86.
96. 97. 98. 99.
106. Recalcati, S. et al. Iron regulatory proteins 1 and 2 in human monocytes, macrophages
and duodenum: expression and regulation in hereditary hemochromatosis and iron
deficiency. Haematologica 91, 303–310 (2006).
Missirlis, F. et al. Compartment-specific protection of iron-sulfur proteins by superoxide dismutase. J. Biol. Chem. 278, 47365–47369 (2003).
Clarke, S.L. et al. Iron-responsive degradation of iron-regulatory protein 1 does not
require the Fe-S cluster. EMBO J. 25, 544–553 (2006).
Wallace, M.A. et al. Superoxide inhibits 4Fe-4S cluster enzymes involved in amino
acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase.
J. Biol. Chem. 279, 32055–32062 (2004).
Brown, N.M., Kennedy, M.C., Antholine, W.E., Eisenstein, R.S. & Walden, W.E.
Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1. Insights into the mechanism of Fe-S cluster cycling.
J. Biol. Chem. 277, 7246–7254 (2002).
Gonzalez, D., Drapier, J.C. & Bouton, C. Endogenous nitration of iron regulatory
protein-1 (IRP-1) in nitric oxide-producing murine macrophages: further insight into
the mechanism of nitration in vivo and its impact on IRP-1 functions. J. Biol. Chem.
279, 43345–43351 (2004).
Pitula, J.S. et al. Selective inhibition of the citrate-to-isocitrate reaction of cytosolic
aconitase by phosphomimetic mutation of serine-711. Proc. Natl. Acad. Sci. USA
101, 10907–10912 (2004).
Fillebeen, C., Caltagirone, A., Martelli, A., Moulis, J.M. & Pantopoulos, K. IRP1 Ser711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase
activities. Biochem. J. 388, 143–150 (2005).
Chen, O.S., Schalinske, K.L. & Eisenstein, R.S. Dietary iron intake modulates the
activity of iron regulatory proteins and the abundance of ferritin and mitochondrial
aconitase in rat liver. J. Nutr. 127, 238–248 (1997).
LaVaute, T. et al. Targeted deletion of iron regulatory protein 2 causes misregulation
of iron metabolism and neurodegenerative disease in mice. Nat. Genet. 27, 209–214
Galy, B. et al. Altered body iron distribution and microcytosis in mice deficient in iron
regulatory protein 2 (IRP2). Blood 106, 2580–2589 (2005).
Meyron-Holtz, E.G. et al. Genetic ablations of iron regulatory proteins 1 and 2 reveal
why iron regulatory protein 2 dominates iron homeostasis. EMBO J. 23, 386–395
Meyron-Holtz, E.G., Ghosh, M.C. & Rouault, T.A. Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo. Science 306, 2087–2090 (2004).
Koh, H.J. et al. Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role
in lipid metabolism. J. Biol. Chem. 279, 39968–39974 (2004).
Lawlis, V.B. & Roche, T.E. Effect of micromolar Ca2+ on NADH inhibition of bovine
kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal
amplification. Mol. Cell. Biochem. 32, 147–152 (1980).
Palmieri, F. et al. Mitochondrial metabolite transporters. Biochim. Biophys. Acta
1275, 127–132 (1996).
Smith, S.R. et al. Severity of neurodegeneration correlates with compromise of iron
metabolism in mice with iron regulatory protein deficiencies. Ann. NY Acad. Sci.
1012, 65–83 (2004).
Zhang, P. et al. Electron tomography of degenerating neurons in mice with abnormal
regulation of iron metabolism. J. Struct. Biol. 150, 144–153 (2005).
Kim, H.Y., Klausner, R.D. & Rouault, T.A. Translational repressor activity is equivalent
and is quantitatively predicted by in vitro RNA binding for two iron-responsive element
binding proteins, IRP1 and IRP2. J. Biol. Chem. 270, 4983–4986 (1995).
Wu, K.J., Polack, A. & Dalla-Favera, R. Coordinated regulation of iron-controlling
genes, H-ferritin and IRP2, by c-MYC. Science 283, 676–679 (1999).
Iwai, K. et al. Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins. Proc. Natl. Acad. Sci.
USA 95, 4924–4928 (1998).
Iwai, K., Klausner, R.D. & Rouault, T.A. Requirements for iron-regulated degradation
of the RNA binding protein, iron regulatory protein 2. EMBO J. 14, 5350–5357
Guo, B., Phillips, J.D., Yu, Y. & Leibold, E.A. Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J. Biol. Chem. 270, 21645–21651
Wang, J. et al. Iron-mediated degradation of IRP2, an unexpected pathway involving a
2-oxoglutarate-dependent oxygenase activity. Mol. Cell. Biol. 24, 954–965 (2004).
Bourdon, E. et al. The role of endogenous heme synthesis and degradation domain
cysteines in cellular iron-dependent degradation of IRP2. Blood Cells Mol. Dis. 31,
Yamanaka, K. et al. Identification of the ubiquitin-protein ligase that recognizes
oxidized IRP2. Nat. Cell Biol. 5, 336–340 (2003).
Jeong, J., Rouault, T.A. & Levine, R.L. Identification of a heme-sensing domain in
iron regulatory protein 2. J. Biol. Chem. 279, 45450–45454 (2004).
Ishikawa, H. et al. Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2. Mol. Cell 19, 171–181 (2005).
Hanson, E.S., Rawlins, M.L. & Leibold, E.A. Oxygen and iron regulation of iron regulatory protein 2. J. Biol. Chem. 278, 40337–40342 (2003).
Schalinske, K.L. & Eisenstein, R.S. Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL60 cells. J. Biol. Chem. 271, 7168–7176 (1996).
Smith, S.R., Ghosh, M.C., Ollivierre-Wilson, H., Hang Tong, W. & Rouault, T.A.
Complete loss of iron regulatory proteins 1 and 2 prevents viability of murine zygotes
beyond the blastocyst stage of embryonic development. Blood Cells Mol. Dis. 36,
283–287 (2006). VOLUME 2 NUMBER 8 AUGUST 2006 NATURE CHEMICAL BIOLOGY ...
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