Download Iron-regulatory proteins, iron-responsive elements and

The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
www.elsevier.com/locate/ijbcb
Review
Iron-regulatory proteins, iron-responsive elements and
ferritin mRNA translation
Andrew M. Thomson a,*, Jack T. Rogers b, Peter J. Leedman a
a
Laboratory for Cancer Medicine and University Department of Medicine, Royal Perth Hospital, GPO Box X2213, Perth, WA 6001,
Australia
b
Division of Hematology-Oncology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
02115, USA
Received 29 March 1999; accepted 30 March 1999
Abstract
Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse
functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell
proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional
regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within
ferritin and transferrin receptor (TfR) mRNAs. The TfR transports iron into cells and the iron is subsequently
stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron
requirements in a coordinate manner by di€erentially regulating ferritin mRNA translational eciency and TfR
mRNA stability. Several di€erent stimuli, as well as intracellular iron levels and oxidative stress, are capable of
regulating these RNA±protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying
modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression. # 1999
Elsevier Science Ltd. All rights reserved.
Keywords: Iron-regulatory proteins; Iron-responsive element; Ferritin; RNA-binding proteins; Posttranscriptional regulation
1. Introduction
Iron is an essential nutrient and a potential
toxin. The cloning of the iron-regulatory proteins
* Corresponding author. Tel.: +61-89-224-0336; fax: 61-89224-0246.
E-mail address: [email protected] (A.M.
Thomson)
(IRPs) and understanding of their interaction
with iron-responsive elements (IREs) has revealed
a fascinating and exquisite system for controlling
iron homeostasis in mammalian cells [1]. In vertebrate species, uptake of transferrin bound iron
into cells occurs via transferrin-receptor (TfR)
mediated endocytosis [2,3]. An uncharacterised
low molecular weight iron chelate mediates distribution of iron within the cell and iron released
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 8 0 - 1
1140
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
Nomenclature
Cys
eALAS
FeATPase
GOX
H 2O 2
IL
IRE
IRP
mt-acon
MAPK
NO
nt
cysteine
erythroid 5-aminolaevulate synthase
iron ATPase
glycolate oxidase
hydrogen peroxide
interleukin
iron-responsive element
iron-regulatory protein
mitochondrial aconitase
mitogen activated protein kinase
nitric oxide
nucleotides
from transferrin is incorporated into newly
formed iron containing proteins and/or the iron
storage protein, ferritin [4±6] (Fig. 1). Iron is
found stored in cells in the ferric state as ferric
oxyhydroxide (Fe(III)OOH) bound to ferritin [6].
Ferritin is a mixture of 24 heavy (H-, 21 kDa)
and light (L-, 19 kDa) chain subunits and these
subunits are coded for by their corresponding
mRNAs [6]. Maintenance of iron homeostasis is
obviously complex and a ®nely tuned system for
the regulation and sensing of cellular iron
requirements is, therefore, paramount to all cells.
Central to this theme in iron homoestasis is
the existence of two mammalian IRPs, IRP1 and
IRP2, whose function is modulated by iron. IRPs
behave as cytoplasmic trans-acting mRNA-binding proteins. At least one type of IRP has been
found in every mammalian cell type studied, and
more recently in the invertebrates, Drosophila
melanogaster and Caenorhabditis elegans[7].
Posttranslational modi®cation of these RBPs
alters their binding anity to their speci®c cognate mRNA hairpin structures, the iron responsive elements (IREs), and controls expression of
target genes posttranscriptionally. IREs present
in the 5'-untranslated region (UTR) of ferritin
high (H) and low (L) chain [6], erythroid 5-aminolaevulinate synthase (eALAS) [8], mammalian
mitochondrial aconitase (mt-acon) [9] and
D.melanogaster succinate dehydrogenase subunit
O2
OH
PKC
REMSA
Ser
Stats
SUS
Thr
TNF
T3
TRH
Superoxide anion
hydoxyl radical
protein kinase C
RNA electrophoretic mobility shift
assay
serine
signal transducers and activators of
transcription
Sequences Upstream of Start codon
threonine
tumour necrosis factor
thyroid hormone
thyrotropin releasing hormone
b [10] mediate the translational eciency of these
mRNAs (Fig. 1). 5 individual IRE motifs present
in the 3'-UTR of the transferrin receptor (TfR)
[11] mediate mRNA stability (Fig. 2). The coordinate but divergent posttranscriptional regulation of IRP/IRE RNA±protein interactions in
these genes governing iron uptake, storage and
usage in cellular iron metabolism maintains intracellular iron at steady state levels (see Figs. 1 and
2).
Much work has focussed on understanding
how the IRP/IRE RNA±protein interaction is
modulated as it forms the foundation of the cellular iron sensory and regulatory network.
Recent evidence suggests that IRP binding is
regulated di€erently in di€erent cell types and
that there are a number of e€ectors that modulate IRP/IRE RNA±protein interactions. Indeed,
if IRPs are central regulators of iron homeostasis
and iron is required for multiple diverse cellular
functions, then responses to multiple e€ectors,
other than iron, should be apparent. Important
modulators of IRP binding and/or function
include nitric oxide (NO) [12], oxidative stress by
H2O2 [13] and hypoxia [14], erythropoietin [15],
hepatic a-antitrypsin [16], phosphorylation by
protein kinase Cs (PKCs) [17], thyroid hormone
(T3) [18] and heme [19]. We have also found that
thyrotropin releasing hormone (TRH) and phosphatases also modulate IRP1 and IRP2 binding
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
1141
Fig. 1. Basic overview of cellular iron metabolism. Transferrin-bound iron is taken into the cell by TfR-mediated endocytosis,
released from the endosome, processed into an inactive form, and sequestered as ferritin or used for immediate metabolic demands,
especially in the mitochondrion. Iron and heme levels can modulate IRP/IRE interactions and coordinately regulate ferritin and
TfR levels. The metabolic byproducts, H2O2/O2/OH, can also alter iron levels and IRP/IRE anity. Extracellular stimuli (e.g.,
erythropoietin, TRH, T3, IL-1b, NO, and H2O2) activate intracellular kinases and phosphatases that modulate IRP phosphorylation status, or cause subsequent redox changes, that alter IRP anity for IRE (AB=acute box sequence).
in pituitary cells. As there is a major review in
this issue (by Ponka and Lok) on the regulation
of TfR gene expression, including the role of
IRPs in controlling TfR mRNA stability, we will
concentrate on the role of IRPs in the translational regulation of ferritin gene expression in
this mini-review.
2. IREs, IRPs and translational control
The IRE stem-loop structure (Fig. 3A) acts as
both a repressor and an enhancer of translation.
The position of the IRE relative to the 5 'UTR
mRNA cap structure (<60 nt) has been shown
to be a crucial factor regulating translation [20].
Binding of an IRP to the IRE is thought to sterically hinder the ability of the 43 S initiation complex (consisting of eIF4E, eIF4G, eIF3 and the
40 S ribosomal subunit) from forming (blocking
recruitment of the 40 S ribosomal subunit to the
mRNA) or from binding at the 5' cap, and so
inhibits initiation of mRNA translation (Fig. 2)
[21,22]. The IRE/IRP interaction is not suciently stable to prevent scanning by the small
ribosome subunit after it has attached to the 5'
cap site, and once translation has been initiated,
the IRE stem-loop is melted by the advancing
43 S scanning ribosomal subunit (Fig. 2). The
IRE sequence is thought to act as a translational
1142
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
Fig. 2. Posttranscriptional regulation of ferritin and TfR mRNAs by IRP1 and 2 interactions with IRE. IRP1 and 2, when bound
to the 5'UTR IRE of ferritin mRNA block the binding of the ribosomal initiation complex and inhibit translation. The binding of
an uncharacterised RNA-binding protein to the acute box (AB) stem loop also plays a role in translational initiation. Both stemloop structures may be important for steric presentation of the mRNA to the initiation complex. (Interaction of poly A tail and associated proteins in translation not shown in this diagram). When IRP1 and 2 are bound to the 5 IREs in the TfR mRNA
3'UTR, they block endonuclease attack which results in an increase in TfR mRNA stability. TRH and T3 can increase or decrease
IRP1 and IRP2 binding activity to the ferritin IRE depending on the cell type.
enhancer region by attracting the intiation complex preferentially to other 5'UTR sequences
[23], though this is still controversial and other
sequences, especially in the 5 'UTR of the H
and L ferritin, may act as the enhancer regions
[24].
The nucleotide sequence of IRE stem-loops is
highly conserved in the genes in which they have
been described within and across species (Fig.
3A) [25]. Elucidation of the structure by mutational analysis and nuclear magnetic resonance
(NMR) indicates that IREs contain 2 regions,
each of which is necessary for IRP binding [26].
The ®rst, a hairpin loop, comprises a 6 nt loop
(5 'C1A2G3U4G5N6) at the end of a ®ve base pair
stem and NMR data suggest important hydrogen
bonding between C1 and G5 in a tertiary interaction across the loop [25]. Mutations within the
®rst 5 nt of the loop reduce IRP binding [25].
Altering the loop sequence in vitro can select for
speci®c binding of IRP1 (UAGUAC) or IRP2
(CCGAGC) [27]. Interestingly, individuals su€ering from familial hyperferritinaemia carry a
C2 4 G2 mutation in the ferritin L-chain gene
which leads to excessive ferritin sysnthesis [28].
The second critical region of the IRE is the contiguous sequence containing either an internal
loop/bulge (found in H- and L-chain ferritin
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
1143
Fig. 3. (A) Predicted structures of identi®ed IREs. Conserved regions of the IRE hairpin-loop (CAGUG) are shown in bold. The
boxed region of the ferritin IRE indicates the speci®c internal loop/bulge which has an increased anity for IRP2 than the bulge
structure of the other IREs. (B) Schematic comaparison of IRP1 and IRP2 sequences. Sequence comparison is high, and the position of the 73-amino acid (aa) IRP2 speci®c region is shown. The cysteine (C) residues important for IRE and iron-sulphur cluster
binding are indicated. Percentages represent the level of identity of each domain of IRP2 compared to IRP1. (C) Postulated model
of IRP1 apoprotein and haloprotein structure. High iron levels induce formation of the iron±sulphur cluster (4Fe±4S) which closes
the putative cleft site and blocks IRE binding but allows citrate/iso-citrate to bind. Low iron levels causes iron±sulphur cluster disassembly which allows IRE entry and which facilitates IRP binding. The cysteine residues considered important in IRE and ironsulphur cluster formation are indicated. IRP1 is synthesised in its apoprotein form. Under certain high iron conditions the 3Fe±4S
form of IRP1 is degraded.
mRNAs) or a cytosine (C)-bulge (TfR, eLAS,
mt-acon mRNAs) [29]. Recently, a thermodynamically unstable IRE-like motif with an A:A mismatch in the contiguous region was found in the
5'-UTR of the murine glycolate oxidase (GOX)
mRNA [30] (Fig. 3A). However, translational
eciency of the GOX mRNA was not controlled
by IRP1 and IRP2 in vivo, although the
sequence bound IRP1 and IRP2 in vitro [30].
IRP1 and IRP2 isoforms bind equally and with
higher anity to the internal loop/bulge structure
(ferritin) than to the C-loop. However, binding
anity of IRP2 to the C-loop is much lower
than for IRP1 [29]. The abundance of the two
IRP isoforms varies considerably in di€erent cell
types and together with di€erential binding to
IREs by IRP1 and IRP2, this system provides
for high precision and ®ne-tuning of IRE-dependent mRNA regulation (see below).
Human IRP2 (105 kDa) has 61% sequence
identity and 79% similarity to human IRP1 and
this degree of homology is conserved across
1144
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
Fig. 4. Demonstration of IRP1 and IRP2 in rat pituitary cells. (A) RNA-gel shift (REMSA) of rat pituitary cytoplasmic protein
extracts from the mammosomatroph GH3 cell line showing IRP1 and IRP2 binding to a 32P-radiolabeled ferritin IRE riboprobe.
The ferritin IRE was incubated with 10 mg GH3 cell cytoplasmic protein extract at 228C for 30 min. RNase T1 (1 U/sample) and
heparin (5 mg/ml ®nal concentration) was then added prior to resolving on a 4.2% PAGE gel. The gel was dried and exposed by
PhosphorImage. The RNA-protein complexes were supershifted with isoform-speci®c antibodies. (B) GH3 cell cytoplasmic protein
extract (20 mg) was incubated with 32P-ferritin IRE probe for 30 min at room temperature as above, RNaseT1 and heparin added,
followed by UV-irradiation for 10 min. The samples were then resolved on a 7.5% SDS±PAGE gel, dried and analysed by
PhosphorImage. This UV-crosslinking analysis (UCA) of a rat pituitary cytoplasmic protein extract shows IRP1 (97 kDa) and
IRP2 (102 kDa).
species. IRP2 (962 amino acids) is notably distinguished from IRP1 (889 amino acids) by a
unique 73 amino acid segment which is responsible for mediating proteolytic cleavage of the
protein in iron replete cells (Fig. 3B). The two
IRPs are readily detectable in rat pituitary tissue
using RNA-electrophoretic mobility shift assays
(REMSA). Fig. 4A shows the IRP1 and IRP2
RNA±protein complexes with a ferritin IRE. In
addition, each IRP was supershifted using isoform-speci®c antibodies. In UV-crosslinking
assays the IRPs migrate at 95±105 kDa (Fig.
4B). Both IRPs comprise a 4 domain structure,
with domain 4 linked (directly to domain 3) by a
hinge region to the other 3 domains (Figs. 3B,
3C) [31]. Most of the recent research on the binding of the IRPs to IREs using site directed mutagenesis, UV-crosslinking and proteolytic analysis
has shown that several regions within the protein
moieties are important in RNA-binding, protein
structural alterations and the modulation of
binding [32,33]. The conservation of binding site
structure helps explain why IREs demonstrate
similar high anity binding to both IRP1 and
IRP2 (Kd 1 10±30 pM) [17]. Cleft exposure is
controlled by the positioning of domain 4 relative
to domains 1±3 (see later). Although IRP1 and
IRP2 are similar proteins that share 61% overall
amino acid identity, they do seem to exhibit
di€erent biochemical properties and mechanisms
of regulation which would allow for precise
changes in the regulation of iron homeostasis
within a cell subjected to many stimuli. It is of
particular interest that the ratios of IRP1:IRP2 is
cell type speci®c (e.g., IRP1>IRP2 in liver, kidney, intestine, brain; IRP1 < IRP2 in pituitary,
Ba/F3 pro-B lymphocyte cell line [34]). This
suggests that IRP2, as well as IRP1, plays an
equally important role in governing iron homeostasis in specifc cell types.
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
3. Regulation of IRP1 binding activity
3.1. Iron induced modulation
IRP1 was the ®rst IRP to be described and,
until recently, was most intensively investigated.
Its central importance in intracellular iron
homeostasis was demonstrated when IRP1 overexpression rendered the cell unable to regulate its
iron levels [35]. IRP1 is a bifunctional protein,
acting either as a cytoplasmic aconitase (haloprotein) in iron replete cells or as a RNA-binding
protein (apoprotein) in iron depleted cells. This
functional switch is dependent upon the assembly
of a iron-sulphur (4Fe±4S) cubane structure in
iron replete cells that is bound by the 3 cysteines
(Cys) in the cleft of the protein [36] and limits
IRE access, but allows the binding of citrate
[33,37]. It is hypothesised that the removal of the
iron±sulphur cluster, a process which is still not
fully understood, causes major structural rearrangements that increase the accessibilty of
IREs to the putative cleft site between domains
1±3 and 4 (Fig. 3C) [38]. Indeed, IRP1 exists in 3
forms within cells; either as a cytoplasmic aconitase haloprotein (4Fe±4S, binds citrate, not an
RNA-binding protein), devoid of aconitase activity (3Fe±4S, binds citrate, not an RNA-binding protein), or as an apoprotein (RNA-binding
protein, little citrate binding). It is not clear at
this point in time whether the 3Fe±4S form represents a physiological intermediate in cluster
assembly/disassembly. However, it is evident that
both functions of IRP1 (aconitase and RNAbinding) are mutually exclusive even though the
putative cleft serves as a common locus for the
two functions of the protein (Fig. 3C) [38].
Speci®c cysteine residues, C437, C503 and
C506, have been identi®ed in the putative cleft of
IRP1 that are critical for the formation of the
stable iron±sulphur cluster and RNA-binding
[36]. The oxidation state of these three residues is
central in the IRP1 response to iron (Figs. 3B
and C) [1]. Several studies have shown that other
regions of IRP1 (amino acids 116±151, 480±623,
and the C-terminal region) also contribute to the
dual functionality of the protein [1,39]. The iron±
sulphur cluster appears to limit the access to po-
1145
tential phosphorylation residues (Ser 138) and
those residues important in RNA-binding located
in and around the putative cleft [17,39].
Disruption of the Fe±S cluster alters structural
con®gurations of both the putative cleft and
hinge linker regions, increasing the accessibility
of the cleft to RNA (Fig. 3C) [39].
There appears to be strict control of the steady
state level of cellular IRP1. As is evident from
above, iron induced alterations in IRP1-binding
activity are due to posttranslational changes in
IRP1 con®guration and are not generally due to
changes in protein levels. However, under certain
conditions when iron enters a cell, IRP1 levels
may be substantially reduced due to degradation,
though this is very controversial [19,40,41]. A
recent report showed that iron chelators and
heme synthesis inhibitors (see Fig. 1) blocked
iron induced IRP1 degradation, but not hemeinduced IRP1 degradation [19]. One report has
shown that IRP1 is dimerised and then degraded
by heme [40], although another report demonstrated that e€ects on IRP1 are by di€erent
forms of iron [41]. Indeed, an earlier work
demonstrated that translational control of
mRNAs coding for H- and L-ferritin was regulated by chelateable intracellular iron pools [42].
The mechanism involved in the postulated hemeinduced degradation of IRP1 is unknown [19].
The pool of IRP1 targetted for degradation is
unclear, but may comprise the subset of proteins
that lack an iron±sulphur cluster [19]. Clearly,
further research is required in this controversial
area.
3.2. Hormone (erythropoietin, TRH, T3) and
kinase induced modulation
Ligands, other than iron, have recently been
shown to alter IRP1 binding activity. Protein
sequence pro®les indicate that Ser 138 and Ser
711 are potential PKC phosphorylation sites in
IRP1. Activation of cellular PKCs with phorbol12 13-myristate acetate (PMA) results in phosphorylation of IRP1 and an increase in binding
to IRE [17,34]. Thus, activation of various signalling pathways that converge on PKC may have
the potential to modify the phosphorylation sta-
1146
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
tus and binding activity of IRP1. Indeed, we
have con®rmed that PKC contributes to basal
levels of IRP1 binding activity. Interestingly,
mitogen activated protein kinases (MAPKs) contribute to maintaining basal IRP1 binding levels.
Furthermore, we have recently demonstrated in
pituitary cells that basal IRP1 binding to a ferritin IRE is regulated by T3 [18] and have subsequently found that TRH also has an e€ect.
This IRP-regulation in the pituitary is accompanied by multiple kinase and phosphatase
activation. This suggests that multiple hormones
have the capacity to regulate IRP1 RNA-binding
by activation of various signalling pathways (Fig.
1). Interestingly, RNA bound IRP1 is less susceptible to phosphorylation by PKC than nonRNA bound IRP1 [17,34]. Phosphorylation of
Ser 138 in IRP1 may serve as a means of partitioning the protein between its two opposing
functions by selectively a€ecting the ability of
residues 116±151 (RNA-binding) and residues
125 and 126 (essential for aconitase function) to
support the separate functions of IRP1. It is hypothesised that phosphorylation may block or
inhibit cluster assembly, inhibiting reversion of
the apoprotein to the haloprotein. This may also
alter the set point at which iron a€ects IRP1
function [17].
Erythropoietin is a kidney produced glycoprotein that enhances red blood cell mass by stimulating proliferation and di€erentiation of near
mature red blood progenitor cells. Erythropoietin
has recently been shown to increase the binding
activity of IRP1 to TfR mRNA speci®cally in
erythroid cells [15], leading to an increase in iron
uptake via an increase in TfR number due to an
increase in TfR mRNA stability. Binding of erythropoietin to its cell surface receptor sets in
motion a cascade of kinase activation, including
MAPKs and PKCs, which are most likely responsible for modulating IRP1 phosphorylation
and RNA-binding activity [43].
3.3. Nitric oxide (NO) induced modulation
An interesting connection between NO and the
IRP has been established [44±46]. NO is a powerful vascular dilator, and much interest surrounds
the possible role of iron in regulating vascular
tone. The observation some years ago that NO
could modulate IRP-binding suggested a plausible connection between the two. NO induces a
slow increase in IRP1 binding activity resulting
in decreased ferritin and increased transferrin
synthesis in hepatic cells [44]. NO activates IRP1
via a cycloheximide-insensitive posttranslational
mechanism [45]. Although the precise mechanisms are unclear, NO may regulate IRP1-binding
through either its ability to disassemble Fe±S
clusters, or, as most recent evidence suggests, to
act as a cytoplasmic iron-chelator thereby limiting intracellular iron availability [45]. The e€ect
is readily detected in adjacent cells due to NO
di€usion [47] and is present in all cell types and
tissues studied to date [44,46]. The e€ect is particularly evident in in¯ammatory conditions in
which bacterial endotoxin and cytokines (IL-1b,
IL-6, TNF-a, INF-g ), induce nitric oxide
synthase isoform-2 (NOS-2) production, leading
to an increase in NO production.
3.4. Oxidative stress, phosphatases, hypoxia and
reoxygenation induced modulation
Oxidative stress and the regulation of iron
metabolism are tightly coupled in bacteria. It is
therefore not surprising to ®nd that oxidative
stress (by H2O2) induces a rapid (<60 min)
increase in IRP1 binding activity in vertebrates,
unlike the slow response (>12 h) observed with
NO [13,46]. There is a corresponding alteration
in ferritin (decrease) and transferrin receptor
(increase) synthesis. This e€ect is due to posttranslational modi®cation of IRP1 and is okadaic
acid sensitive [48]. The current hypothesis
suggests phosphatases are involved in the signalling mechanism, being recruited by H2O2, and
acting through a plasma-membrane associated
H2O2 sensor [13]. Interestingly, a recent study
[14] has shown that hypoxia leads to the inactivation of IRP1 binding in hepatoma cells, without any changes in IRP1 protein levels. This
e€ect was reversible on subsequent exposure of
the cells to oxygen. The mechanism governing
this IRP1 modulation is unknown but is thought
to be a phosphatase and/or oxygen radical-
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
dependent [14] posttranslational event, mimicking
the mechanism involved in the H2O2-induced
modulation of IRP1.
4. Regulation of IRP2 binding activity
4.1. Iron induced modulation
The discovery of a second vertebrate IRP,
IRP2, led to the postulate that two IRPs are
essential for high ®delity control of iron homeostasis in cells that are exposed to multiple internal and external stimuli [47,49±51]. As a
consequence, there has been a resurgence of
interest in understanding the mechanisms controlling the regulation of IRP2 in mammalian
cells in the last few years. Indeed, recent studies
have demonstrated the important role it plays in
regulating iron homeostasis. Most signi®cantly,
IRP2 was shown to be resposible for maintaining
normal cellular function and iron homeostasis in
Ba/F3 prolymphocytes which lack IRP1 [39].
IRP2, unlike IRP1, does not form an iron±sulphur cluster in the putative cleft, although the location of the 3 cysteines (C512, C578, C581) is
conserved
when
compared
with
IRP1.
Furthermore, IRP2 does not exhibit any aconitase activity, due to the lack of conservation of
certain amino acids that play a crucial role in
aconitase enzymatic function [47,49±51]. As mentioned earlier, another major di€erence between
IPR2 and IRP1 is the 73 amino acid insertion in
IRP2 that is involved in binding iron and is the
target for a cytoplasmic protease that mediates
IRP2 degradation [52±54].
Variations in iron concentration has little e€ect
on IRP2 transcription. However, IRP2 protein is
degraded eciently by the proteosome in iron-replete cells [52±54], whilst its degradation rate is
decreased in iron-deplete cells [52±54]. IRP2
undergoes an iron-dependent oxidative modi®cation in iron-replete cells that increases degradation by the proteosome by a ubiquitinmediated pathway [52±54]. Mutations of the
cysteine residues, C168 and C201, within the
unique 73 amino acid region eliminates the ability of this sequence to mediate iron-dependent
1147
degradation [52]. It is hypothesised that these
cysteines are involved in the ligation of an iron±
sulphur cluster, and so act as an iron sensor [52].
Free iron is not necessary for the ubiquitination
steps once the protein has been oxidised [53]. The
degradation process may also be induced by
heme levels, similar to IRP1 degradation, rather
than a direct e€ect of iron [19]. It is now clear
that the two cysteine residues, C168 and C201,
within the unique 73 amino acid region of IRP2
that are predicted to be especially redox active
[17,39], are indispensable for iron induced degradation.
4.2. Hormone (erythropoietin, TRH, T3) and
kinase induced modulation
Similar to IRP1, the RNA-binding activity of
IRP2 can be upregulated by phosphorylation via
activation of PKCs [17,34,39]. This e€ect does
not require a change in IRP2 protein level
[17,34,39]. Interestingly, the putative PKC phosphorylation sites of IRP2 di€er to those of IRP1
[17,39]. In addition, putative PKC and MAPK
phosphorylation sites have been found in the
unique 73 amino acid region of IRP2.
Phosphorylation of speci®c serine and threonine
residues in this region may a€ect the redox properties of the nearby cysteine residues that contribute to the redox and iron regulation of IRP2
[39]. Somewhat unexpectedly, the basal level of
phosphorylation of IRP2 is higher than IRP1 in
HL-60 cells [39]. The observation that IRP2
binding may be regulated by phosphorylation
without changes in IRP2 protein levels provides
a second level of regulation. IRP2 binding activity increases in erythropoietin stimulated cells
[15], and this is most likely due to changes in
phosphorylation status of the IRP induced by activation of PKCs and MAPKs. In our studies to
investigate the regulation of gene expression in
the pituitary, we recently found that IRP2 predominates (see Fig. 4A). The binding activity of
IRP2 is regulated by several di€erent stimuli.
These include TRH and T3 which appear to
modulate the activation of PKCs, MAPKs, and
phosphatases, resulting in a change in phosphorylation of IRP2, without changes in IRP2
1148
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
protein levels (data not shown). These data and
those of Schalinske et al. [34] support the concept
that IRP2 may have an important e€ect on iron
homeostasis in speci®c cells and may predominate over IRP1.
4.3. Nitric oxide (NO) induced modulation
The e€ect of NO on IRP2 RNA-binding activity remains controversial. The binding activity
of IRP2 is increased, as with IRP1, after NO
stimulation [45], though this appears to be cell
speci®c [12]. One hypothesis suggests that NO
limits the availabilty of cytoplasmic iron, thereby
reducing binding of iron to the 73 amino acid
insertion and oxidation of IRP2, as well as
degradation by the proteasome [54]. In those
cells where NO increases IRP2 binding activity, it
is slow (>12 h) and appears to be due to an
increase in de novo IRP2 synthesis [45]. A major
drawback in all of these studies to date relates to
the use of cells in which IRP2 is not the predominant isoform. Thus, the e€ect may be di€erent in
cells, such as the pituitary, and remains to be
investigated.
4.4. Oxidative stress, phosphatases, hypoxia and
reoxygenation induced modulation
Despite extensive studies, H2O2 does not
appear to modify IRP2 binding activity
[1,13,45,47]. This is consistent with work demonstrating no e€ect of hypoxia and reoxygenation
on IRP2 binding activity [10]. Again these studies
have su€ered from using cells with concentrations of IRP1 greater than IRP2. However,
these data are consistent with other stimuli such
as phosphatases and kinases being the major
modulators of IRP2 binding action.
In sum, it is evident that a more detailed study
of the mechanisms of IRP2 modulation are
required to fully understand the regulation of
RNA-binding of this important protein.
However, our data and that of others demonstrate that IRP2 can be regulated by many stimuli and that the regulation of IRP2 expression is
complex and di€ers signi®cantly from that of
IRP1. These di€erences between IRP1 and IRP2
regulation provide a far greater level of complexity for controlling intracellular iron homeostasis
than previously envisaged. How each is di€erentially regulated, and the contribution of each in
speci®c cell types is the focus of current work in
this area.
5. Invertebrate IRP1-isoforms
Interestingly, IRP1-isoforms, but no IRP2-isoforms, have recently been identi®ed in
D.melanogaster and C.elegans[7]. One IRP was
found in C.elegans and two IRPs, IRP1A and
IRP1B (86% identical to each other), in
D.melanogaster. IRP1A has been localised to
position 94C1-8 and IRP1B to position 86B3-6
on the right arm of chromosome 3 and both
show 67% homology to IRP1. Human IRP1 has
been localised to human chromosome 9 [55]. The
invertebrate IRPs do not contain an IRP2-like
amino acid insertion, but the aconitase active site
residues are of IRP1-type [7]. It is postulated that
the D.melaonogaster and vertebrate IRPs resulted
from an IRP gene duplication event, and that
this may have occurred prior to the diversion of
invertebrates and vertebrates [7]. In contrast to
vertebrates, in which insertion of a 73 amino acid
coding exon produces IRP2, evolutionary pressures may have been such that further changes to
Drosophila IRPs were not essential. Intriguingly,
IRP1A and IRP1B are not di€erentially
expressed in speci®c organs, unlike mammalian
IRP1 and IRP2 [7]. However, the fact that invertebrates evolved a second IRP, albeit another
IRP1 derivative, reinforces the notion that two
IRPs are essential for high ®delity control of iron
homeostasis in cells exposed to multiple internal
and external stimuli.
It is apparent from all the above that the IRP/
IRE interactions play a pivotal role in controlling
iron homeostasis in invertebrates and vertebrates
via regulation of ferritin gene expression.
However, cis-elements, other than IREs, have
recently been identi®ed that have a major impact
on ferritin gene expression.
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
6. Non-IRP/IRE regulation of ferritin translation
Ð the acute box
There has long been a clinical association
between serum iron levels, serum ferritin levels,
intracellular ferritin levels and cytokine induced
in¯ammation [56]. Indeed, elevated serum ferritin
levels have been used as a diagnostic for in¯ammation and disease. IL-1 is the major in¯ammatory cytokine that lowers blood serum iron levels
[57], and an increase in ferritin synthesis immediately precedes a lowering in serum iron levels
[58]. The elevated ferritin levels lead to a lowering of serum iron levels through iron sequestration. IL-1 induces an increase in hepatic ferritin
synthesis by increasing ferritin mRNA translation
in hepatic cells [59]. This IL-1 induced response
increases serum ferritin levels due to tissue export
of ferritin to the bloodstream [28]. This increase
in tissue and serum ferritin levels also occurs in
hereditary cataract hyperferritinemia syndrome,
in which an IRE mutation in the L-ferritin gene
reduces IRP binding and leads to uncontrolled
L-ferritin synthesis [28,60]. Remarkably, a 20 nt
acute box cis-element downstream of the IRE in
the 5 '-UTR of H- and L-ferritin mRNAs was
recently identi®ed (Figs. 1 and 2) which mediates
this IL-1 e€ect on ferritin translation in both
hepatic and endothelial cells [61,24].
The importance of the acute box as a major
regulator of ferritin synthesis was established in
experiments in which mutations of the 5'-UTR
sequence resulted in an eightfold reduction in the
level of ferritin synthesis [24]. The mutations may
change the steric con®guration of the 5'-UTR
and reduce the mRNA anity to the translational pre-initiation complex. Recent investigations have found a trans-acting RNA-binding
protein in hepatic cells that binds to the acute
box cis-element in H- and L-ferritin mRNAs
(J.T. Rogers, unpublished results), and mutations
in the acute box may alter binding anity of this
RNA-binding protein. Our current hypothesis is
that during in¯ammation ferritin translation is
primarily regulated by IL-1 modulation of binding of this RBP to the ferritin mRNA 5'-UTR
acute box. The acute box also plays an important
role in the regulation of ferritin synthesis in pitu-
1149
itary lactotroph cells stimulated with TRH and
T3 in which there is reduced IRP/IRE binding,
but a decrease in ferritin synthesis (A.M.
Thomson, J.T. Rogers and P.J. Leedman, unpublished results). Thus, evidence is evolving to indicate that the posttranscriptional regulation of
ferritin levels involves at least two cis-acting
mRNA elements and two sets of RBPs, which
may act together or independently and which
may be controlled by multiple stimuli.
7. Concluding remarks
Vertebrates and invertebrates have devised elaborate and exquisitely controlled systems to
regulate iron homeostasis. The IRPs, which are
conserved across species, play a central role as
bifunctional regulators of ferritin mRNA translational eciency and TfR mRNA stability. Acting
in concert, these IRP/IRE interactions are a
superb example of how cells have crafted a master control system based on a single high anity
RNA±protein interaction that has great complexity, due to the expanding set of compounds that
regulate the interaction. The discovery of reciprocal and coordinate regulation of IRP/IRE interactions and establishing their role in modulating
iron homeostasis represent major discoveries in
the ®eld. At the molecular level, the interplay
between IRPs and IREs of ferritin and transferrin receptor mRNA have formed the basis of
much of our understanding of RNA±protein interactions and posttranscriptional regulation of
genes. De®ning the mechanisms controlling ferritin translation represents a major advance in
understanding
translational
initiation.
Elucidation of the regulation of TfR mRNA
stability has provided great insights into the regulation of mRNA turnover. The discovery of a
second IRP has presented many challenges to de®ne the role of each in what appears to be a cellspeci®c set of regulatory events. It has also provided the framework for extremely ®ne tuning of
intracellular iron homeostasis through di€erential
expression of the isoforms. The ubiquitination
and subsequent degradation by the proteasome
of oxidised IRP2 has added to the knowledge of
1150
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
cellular control of oxidatively damaged cytoplasmic proteins. Appreciation of the role of the
acute box in regulating ferritin synthesis has
emphasised that posttranscriptional regulation of
mRNAs is likely to involve the interaction of
multiple RNA-binding proteins and their cognate
cis-acting mRNA sequences. The last decade has
seen an explosion of information in the biology
of the IRPs. We look forward to future work
which will unravel some of the mysteries of the
functional roles of IRP1 and IRP2, identify novel
regulators of IRP1 and IRP2 activity, clarify the
interaction of IRPs with other RNA-binding proteins in the regulation of iron homeostasis, and
solve the enigma of IRP1 having aconitase activity.
[10]
[11]
[12]
[13]
[14]
References
[1] M.W. Hentze, L.C. KuÈhn, Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress,
Proceedings of the National Academy of Sciences USA
93 (1996) 8175±8182.
[2] K. Miller, M. Shipman, I.S. Trowbridge, C.R. Hopkins,
Transferrin receptors promote formation of clathrin lattices, Cell 65 (1991) 621±631.
[3] N.C. Andrews, J. Levy, Iron is hot: an update on the
pathophysiology of hemachromatosis, Blood 92 (1998)
1845±1851.
[4] D.M. De Silva, S.D. Aust, Ferritin and ceruloplasmin in
oxidative damage: review and recent ®ndings, Canadian
Journal of Physiology and Pharmacology 71 (1993)
715±720.
[5] P. Ponka, Tissue speci®c regulation of iron metabolism
and heme synthesis: distinct control mechanisms in erythroid cells, Blood 89 (1997) 1±25.
[6] E.C. Theil, Iron-responsive element (IRE) family of
mRNA regulators. Regulation of iron transport and
uptake in animals, plants and microorganisms, Metal
Ions in Biological Systems 35 (1998) 403±434.
[7] M. Muckenthaler, N. Gunkel, D. Frishman, A.
Cyrkla€, P. Tomancak, M.W. Hentze, Iron-regulatory
protein-1 (IRP-1) is highly conserved in two invertebrate
species. Characterisation of IRP-1 homologues in
Drosophila melanogaster and Caenorhabditis elegans,
European Journal of Biochemistry 254 (1998) 230±237.
[8] T.C. Cox, M.J. Bawden, A. Martin, B.K. May, Human
erythroid 5-aminolevulinate synthase: promotor analysis
and identi®cation of an iron-responsive element in the
mRNA, The EMBO Journal 10 (1991) 1891±1892.
[9] M.K. Gray, K. Pantopoulos, T. Dandekar, B.A.C.
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
Ackrell, M.W. Hentze, Translational regulation of
mammalian and Drosophila citric acid cycle enzymes via
iron-responsive elements, Proceedings of the National
Academy of Sciences USA 93 (1996) 4925±4930.
S.A. Kohler, B.R. Henderson, L.C. KuÈhn, Succinate dehydrogenase b mRNA of Drosophila melanogaster has a
functional iron-responsive element in its 5'-untranslated
region, The Journal of Biological Chemistry 270 (1995)
30,781±30,786.
R.D. Klausner, T.A. Rouault, J.B. Harford, Regulating
the fate of mRNA: the control of cellular iron metabolism, Cell 72 (1993) 19±28.
J.D. Phillips, D.V. Kinikini, Y. Yu, B. Guo, E.A.
Leibold, Di€erential regulation of IRP1 and IRP2 by
nitric oxide in rat hepatoma cells, Blood 87 (1996)
2983±2992.
K. Pantopoulos, M.W. Hentze, Activation of iron regulatory protein-1 by oxidative stress in vitro, Proceedings
of the National Academy of Sciences USA 95 (1998)
10,559±10,563.
E.S. Hanson, E.A. Leibold, Regulation of iron regulatory protein 1 during hypoxia and hypoxia/reoxygenation, The Journal of Biological Chemistry 273 (1998)
7588±7593.
G. Weiss, T. Houston, S. Kastner, K. JoÈhrer, K.
GruÈnewald, J.H. Brock, Regulation of cellular iron
metabolism by erythropoietin: activation of iron-regulatory protein and upregulation of transferrin receptor expression in erythroid cells, Blood 89 (1997) 680±687.
I. Graziadei, G. Weiss, A. Bohm, G. Werner-Felmayer,
W. Vogel, Unidirectional upregulation of the synthesis
of the major iron proteins, transferrin-receptor and ferritin, in HepG2 cells by the acute-phase protein a1-antitrypsin, Journal of Hepatology 27 (1997) 716±725.
K.L. Schalinske, R.S. Eisenstein, Phosphorylation and
activation of both iron regulatory proteins 1 and 2 in
HL-60 cells, The Journal of Biological Chemistry 271
(1996) 7168±7176.
P.J. Leedman, A.R. Stein, W.W. Chin, J.T. Rogers,
Thyroid hormone modulates the interaction between
iron regulatory proteins and the ferritin mRNA iron-responsive element, The Journal of Biological Chemistry
271 (1996) 12,017±12,023.
L.S. Goessling, D.P. Mascotti, R.E. Thach, Involvement
of heme in the degradation of iron regulatory protein 2,
The Journal of Biological Chemistry 273 (1998) 12,555±
12,557.
B. Goossen, M.W. Hentze, Position is the determinant
for function of the iron-responsive elements as translational enhancers, Molecular and Cellular Biology 12
(1992) 1959±1966.
A.B. Sachs, P. Sarnow, M.W. Hentze, Starting at the
beginning, middle, and end: translation initiation in
eukaryotes, Cell 89 (1997) 831±838.
M. Muckenthaler, N.K. Gray, M.W. Hentze, IRP1
binding to ferritin mRNA prevents the recruitment of
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
small ribosomal subunit by the cap-binding complex,
Molecular Cell 2 (1998) 383±388.
E. De Gregorio, T. Preiss, M.W. Hentze, Translational
activation of uncapped mRNAs by the central part of
human eIF4G is 5 ' end-dependent, RNA 4 (1998) 828±
836.
J.T. Rogers, Ferritin translation by interleukin-1 and
interleukin-6: the role of sequences upstream of the start
codons of the heavy and light subunit genes, Blood 87
(1996) 2525±2537.
L.G. Laing, K.B. Hall, A model of the iron responsive
element RNA hairpin loop structure determined from
NMR and thermodynamic data, Biochemistry 35 (1996)
13,586±13,596.
B.R. Henderson, E. Menotti, C. Bonnard, L.C. KuÈhn,
Iron-regulatory proteins 1 and 2 bind distinct sets of
RNA target sequences, The Journal of Biological
Chemistry 271 (1996) 4900±4908.
E. Menotti, B.R. Henderson, L.C. KuÈhn, Translational
regulation of mRNAs with distinct IRE sequences by
iron regulatory proteins 1 and 2, The Journal of
Biological Chemistry 273 (1998) 1821±1824.
D. Girelli, R. Corrocher, L. Bisceglia, O. Olivieri, L. De
Franceschi, L. Zelante, P. Gasparini, Molecular basis
for recently described hereditary hyperferritinemia-cataract syndrome: a mutation in the iron-responsive element
of ferritin 1-subunit gene (the ``Verona Mutation''),
Blood 86 (1995) 4050±4053.
Y. Ke, J. Wu, E.A. Leibold, W.E. Walden, E. Theil,
Loops and bulge/loops in iron-responsive element isoforms in¯uence iron regulatory protein binding, The
Journal of Biological Chemistry 273 (1998) 23,637±
23,640.
S.A. Kohler, E. Menotti, L.C. KuÈhn, Molecular cloning
of mouse glycolate oxidase, The Journal of Biological
Chemistry 274 (1999) 2401±2407.
M.J. Gruer, P.J. Artymiuk, J.R. Guest, The aconitase
family: three structural variations on a common theme,
Trends in Biochemistry 22 (1997) 3±6.
E. Paraskeva, M.W. Hentze, Iron-sulphur clusters as
genetic regulatory switches: the bifunctional iron regulatory protein-1, FEBS Letters 389 (1996) 40±43.
J.P. Basilion, T.A. Rouault, C.M. Massinople, R.D.
Klausner, W.H. Burgess, The iron-responsive elementbinding protein: localisation of the RNA-binding site to
the aconitase active-site cleft, Proceedings of the
National Academy of Sciences USA 91 (1994) 574±578.
K.L. Schalinske, K. Blemings, D.W. Ste€en, O.S. Chen,
R. Eisenstein, Iron-regulatory protein 1 is not required
for the modulation of ferritin and transferrin receptor
expression by iron in a murine pro-B lymphocyte cell
line, Proceedings of the National Academy of Sciences
USA 94 (1997) 10,681±10,686.
P.A. DeRusso, C.C. Philpott, K. Iwai, H.S. Mostowski,
R.D. Klausner, T.A. Rouault, Expression of a constitutive mutant of iron-regulatory protein 1 abolishes iron
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
1151
homeostasis in mammalian cells, The Journal of
Biological Chemistry 270 (1995) 15,451±15,454.
H. Hirling, B.R. Henderson, L.C. KuÈhn, Mutational
analysis of the [4Fe-4S]-cluster converting iron-regulatory factor from its RNA-binding form to cytoplasmic
aconitase, The EMBO Journal 13 (1994) 453±461.
A. Constable, S. Quick, N.K. Gray, M.W. Hentze,
Modulation of the RNA-binding activity of a regulatory
protein by iron in vitro: switching between enzymatic
and genetic function?, Proceedings of the National
Academy of Sciences USA 89 (1992) 4554±4558.
D.J. Haile, T.A. Rouault, J.B. Harford, M.C. Kennedy,
G.A. Blondin, H. Beinert, R.D. Klausner, Cellular regulation of the iron-responsive element binding protein:
disassembly of the cubane iron-sulfur cluster results in
high-anity RNA binding, Proceedings of the National
Academy of Sciences USA 89 (1992) 11,735±11,739.
K.L. Schalinske, S.A. Anderson, P.T. Tuazon, O.S.
Chen, M.C. Kennedy, R.S. Eisenstein, The iron-sulfur
cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites,
Biochemistry 36 (1997) 3950±3958.
J-J. Lin, M. Patino, L. Gaeld, W.E. Walsen, A.
Smith, R. Thach, Crosslinking of hemin to a speci®c
site on the 90 kD ferritin repressor protein, Proceedings
of the National Academy of Sciences USA 88 (1991)
6068±6071.
R.S. Eisenstein, D. Garcia-Mayol, W. Pettingell, H.N.
Munro, Regulation of ferritin and heme oxygenase synthesis in rat ®broblasts by di€ereny forms of iron,
Proceedings of the National Academy of Sciences USA
88 (1991) 688±692.
J.T. Rogers, H.N. Munro, Translational control of
mRNAs coding for both heavy and light subunits of ferritin in rat hepatoma cells is regulated by chelateable intracellular iron pools, Proceedings of the National
Academy of Sciences USA 84 (1987) 2277±2281.
M.O. Showers, A.D. D'Andrea, Subunit structure and
transmembrane signalling of the erythropoietin receptor,
International Review of Cytology 137B (1992) 99±120.
J.B. Domachowske, The role of nitric oxide in the regulation of cellular iron metabolism, Biochemical and
Molecular Medicine 60 (1997) 1±7.
K. Pantopoulos, G. Weiss, M.W. Hentze, Nitric oxide
and oxidative stress (H2O2) control mammalian iron
metabolism by di€erent pathways, Molecular and
Cellular Biology 16 (1996) 3781±3788.
G. Weiss, B. Goosen, W. Doppler, D. Fuchs, K.
Pantopoulos, G. Werner-Felmayer, H. Wachter, M.W.
Hentze, Translational regulation via the iron-responsive
elements by the nitric oxide/NO-synthase pathway, The
EMBO Journal 12 (1993) 3651±3657.
L.C. KuÈhn, Iron and gene expression: molecular mechanisms regulating cellular iron homeostasis, Nutrition
Reviews 56 (1998) S11±S19.
K. Pantopoulos, M.W. Hentze, Rapid responses to oxi-
1152
[49]
[50]
[51]
[52]
[53]
[54]
[55]
A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152
dative stress mediated by iron-regulatory protein, The
EMBO Journal 14 (1995) 2917±2924.
B. Guo, Y. Yu, E. Leibold, Iron regulates cytoplasmic
levels of a novel iron-responsive element binding protein
without aconitase activity, The Journal of Biological
Chemistry 269 (1994) 24,252±24,260.
B.R. Henderson, C. Seiser, L.C. KuÈhn, Characterisation
of a second RNA-binding protein in rodents with speci®city for iron responsive elements, The Journal of
Biological Chemistry 268 (1993) 27,327±27,334.
F. Samaniego, J. Chin, K. Iwai, T.A. Roualt, R.D.
Klausner, Molecular characterisation of a second ironresponsive element binding protein, iron-regulatory protein 2, The Journal of Biological Chemistry 269 (1994)
30,904±30,910.
K. Iwai, R.D. Klausner, T.A. Rouault, Requirements
for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2, The EMBO Journal 14
(1995) 5350±5357.
K. Iwai, S.K. Drake, N.B. Wehr, A.M. Weissman, T.
LaVaute, N. Minato, R.D. Klausner, R.L. Devine, T.A.
Rouault, Iron-dependent oxidation, ubiquitination, and
degradation of iron regulatory protein 2: implications
for degradation of oxidised proteins, Proceedings of the
National Academy of Sciences USA 95 (1998) 4924±
4928.
B. Guo, J.D. Phillips, T. Yu, E.A. Leibold, Iron regulates the intracellular degradation of iron-regulatory
protein 2 by the proteosome, The Journal of Biological
Chemistry 270 (1995) 21,645±21,651.
M.W. Hentze, H.N. Seuanez, S.J. O'Brien, J.B.
[56]
[57]
[58]
[59]
[60]
[61]
Harford, R.D. Klausner, Chromosomal localisation of
nucleic acid-binding proteins by anity mapping:
assignment of the IRE-binding protein gene to human
chromosome 9, Nucleic Acids Research 17 (1989) 6103±
6108.
J.T. Rogers, Genetic regulation of the iron transport
and storage genes: links with acute phase response, in:
R.B. Lau€er (Ed.), Iron and human disease, CRC Press,
Boca Raton, 1992, pp. 78±104.
V.R. Gordeuk, P. Prithviraj, T. Dolinar, G.M.
Brittenham, Interleukin-1 administration to mice produces hypoferremia despite neutropenia, Journal of
Clinical Investigation 82 (1988) 1934±1938.
A.M. Konijn, Iron metabolism in in¯ammation,
Baillieres Clinical Haematology 7 (1994) 829±849.
J.T. Rogers, K.R. Bridges, G.P. Durmowicz, J. Glass,
P.E. Auron, H.N. Munro, Translational control during
the acute phase response: ferritin synthesis in response
to interleukin-1, The Journal of Biological Chemistry
265 (1990) 14,572±14,578.
C. Beaumont, P. Leneuve, P. Devaux, I. Devaux, J.Y.
Scoazec, M. Berthier, M-N. Loiseux, B. Grandchamp,
D. Bonneau, Mutation in the iron-responsive element of
the L-ferritin mRNA in a family with dominant hyperferritenaemia and cataract, Nature Genetics 11 (1995)
444±446.
J.T. Rogers, J. Andriotakis, L. Lacroix, K. Kasschau,
G. Durmowicz, K. Bridges, Translational enhancement
of H-ferritin mRNA by interleukin-1b acts through 5'leader sequences distinct from the iron-responsive element, Nucleic Acids Research 22 (1994) 2678±2686.