- Email: [email protected]
Iron metabolism
200
Iron metabolism Philip Aisen *, Marianne The
understanding
of iron metabolism
has been enormously expanded findings about the functioning receptor discovery previously
proteins
at the molecular
in recent of transferrin,
and ferritin. Other recent of the hemochromatosis unknown
Wessling-Resnickt
developments include gene HFE, identification
involved
in iron transport,
metal transporter expanded insights
1 and stimulator of Fe transport, and into the regulation and expression of
proteins
involved
in iron metabolism.
principal although
participants in iron transport have the complexity of such interactions
understood. concepts biology continue
Interactions
Correlated efforts involving of crystallography, spectroscopy applied
to cellular
to be, particularly
processes
level
years by new the transferrin the of divalent
among
been uncovered, is still incompletely
techniques and and molecular have
been,
and should
Opinion
in Chemical
Biology
1999,
3:200-206
http://biomednet.com/elecref/1367593100300200 0 Elsevier
Science
Ltd ISSN
and cloned [2’,3’]. Another protein functioning asa stimulator of iron transport, stimulator of Fe transport (SFT), has been identified and cloned [4]. New insights into the function, regulation and interactions of major proteins of iron metabolism, including transferrin, the transferrin receptor and ferritin, have similarly expanded. Nevertheless, fine details of molecular mechanismsinvolved in iron transport and storageoften remain elusive. In this brief review, we will focus on recent developments in the broad area of iron metabolism-proteins of iron metabolism,cellular transport of iron, and the regulation of iron metabolism- emphasizing uncertainties to be resolved.
Proteins
of iron metabolism
Transferrin
revealing.
Addresses *Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; e-mail: [email protected] Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115-6021, USA; e-mail: [email protected] IEccles Program in Human Molecular Biology and Genetics and the Department of Medicine, University of Utah, Building 533, Room 4220, Salt Lake City, UT 84112, USA; e-mail: [email protected] Current
and Elizabeth A Leiboldl
Transferrin, the iron-transporting plasma protein, provides most of the iron for the physiological needs of iron-requiring cells, and is normally the only known source of iron for hemoglobin synthesis. The detailed structure of the serum transferrin molecule, revealed by X-ray crystallography, shows it to be, as predicted from internal homology, a bilobal protein with each lobe (designated the N and C lobes for their respective sequences in the transferrin molecule) bearing a single metal-binding site and comprising two domains [5,6]. The iron ligands of all known transferrins are two tyrosine residues,a histidine, aspartate and a synergistic carbonate anion linked to both metal and protein. Without such a linking anion, the iron-binding function of transferrin is lost.
1367-5931
Abbreviations DMTl divalent metal transporter H heavy/heart HFE hemochromatosis gene IRE iron-responsive element iron regulatory protein IRP L light/liver m mitochondrial nitric oxide radical NO stimulator of Fe transport SFT UTR untranslated region
1
Introduction In the past few years, our understandingof iron metabolism in unicellular and multicellular organisms ranging from microbes to man has advanced dramatically. HFE, the long sought-after gene responsiblefor hereditary hemochromatosis, an iron-overloading disorder that, untreated, leads to disability and death, hasbeen identified and cloned [ 11,and interactions of the HFE protein with other participants in iron metabolism is under active study. A protein implicated to be a principal iron transporter in intestinal mucosalcells and to alsobe involved in intracellular transmembranetransport, divalent metal transporter 1 (DMTl) (formerly natural resistance-associatedmacrophage protein 2 [NrampZ] or divalent cation transporter 1 [DCTl]), has been identified
In addition to the role of these iron ligands in iron binding and release, the protein fabric itself strongly regulates release. Iron release is generally more facile from the N lobe, particularly at the low pH (near 5.6) of the endosome,where iron is releasedwithin the cell. Protonation of two lysine residues, neighbors in space but situated on opposite domains, results in electrostatic repulsion of the domains as a dominant event in iron release from the N lobe [7,8] but not in the C lobe, which lacks the dilysine pair. A variety of site-directed mutagenesisstudies indicate that residuesnot directly involved in iron binding alsofunction in modulating iron release,and that such outer-shell or remote functions may differ in the two lobes [9’,10’]. Mechanisms of iron binding have received relatively little attention. Most studies suggest that binding of the synergistic anion precedes iron binding [ 111,and that a seriesof deprotonation reactions results in a closing of the two domains of the binding lobe to lock iron into place [12]. A protein with remarkable similarity to transferrin in its iron-binding properties, but without sequence homology to transferrin, has been isolated from the periplasmic space of pathogenic ZV&~~etiia [13’]. Thought to act as an iron
Iron metabolism
;axxeppx fKm hiw L Llalls -- - F-ddl, --* XiEs3SjX3Skiig wx~ss the pei$asmic space to rhe cyK3ph~m
ihC
S-Si>X
af the
Aisen,
Wessling-Resnick
and Leibold
is a homndimetic
g3ycqmkm,
201
&ii
infec-
The
rransferfm
receptor
k3
tious organism, the protein resembles transferrin in displaying two ryrosine ljgands lbur oifiesent>y disposed from the arrangement in transferrin) and one histidine lig-
two subunits linked by a pair of disulfide bonds at or near the inrramembranous porrjon of the mo)ecuk. Of she four glycosylation sites, three are N-linked and one is O-linked;
and, but wirh ghtamak in place of aspar~ate. A partic&dy intriguing finding is the presenceof a noncovalently bound
N-linked &king
phosphate group, apparently acting as the synergistic anion. Whether the bacterial protein will also accommodate carbonate remains to be determined. Ferritin
Ferritin is involved in iron storage and detoxification in microbial, plant and animal species.The mammalianferritin molecule is a heteropolymer of 24 subunits of two types, designated H (for heart or heavy) and L (for liver or light), depending on the organs in which they predominate and their relative molecular weights (typically 19,000-21,000). Sequence identity between H and I, chains is >SO%;the ratio of H to L subunits is a function of the tissue of origin. Subunits can spontaneouslyassembleinto a hollow spherical protein, the interior of which accommodates4,000 or more iron atoms asferrihydrite, FeOOH, although most isolated ferritins contain closer to 2,000 iron atoms. Four hydrophilic channelsof threefold symmetry, and three fourfold hydrophobic channelspierce the protein shell. A widely accepted but still disputed model identifies sites present only within H chains where a combination of glutamate, tyrosine and histidine residues form a ferroxidase center that functions to bind Fe(I1) dimers and catalyze their oxidation via a peroxodiferric intermediate (that was observed in freeze-quench Mossbauer spectroscopy) [14]. Fe(II1) is then conveyed through undefined intrasubunit routes and the hydrophilic channels to the protein cavity, where core formation initiates at carboxyl groups presented by L chain glutamate residues [15] to complete the three-stage process of Fe(II) binding, oxidation and core formation. Iron uptake appears to be driven by electrostatic gradients [16] and facilitated by plasticity in protein conformation, creating pathways (not evident in the crystal structure) for iron to follow. Once core formation is underway the growing core serves to promote iron oxidation, with a 1:4 stoichiometry of dioxygen consumed to iron oxidized, rather than 1~2,as seen in ferroxidase site oxidation [17’]. The alternative view is that Fe(I1) oxidation is carried out not by ferritin but by ceruloplasmin, a multicopper-containing plasmaprotein with known ferroxidase activity, or an intracellular analog of ceruloplasmin [ 181.The resulting Fe(II1) is then transported to the ferritin cavity for mineralization into the core. The controversy continues.
has a cfi&d
The hemochromatosis
de
in fdbing
and craf-
gene
and its product
has been identified, cloned and expressed, the missense mutation responsible for most cases of hemochromatosisfound and the structure of its product determined by X-ray crystallography [1,22”]. The HFE protein is a 343-residue transmembrane glycoprotein homologous to class I major histocompatibility complex (MHC) proteins. Like other MHC proteins, it associates with &-microglobulin at the cell surface, with disruption of such associationleading to generalized iron overload [23]. HFE also associateswith the transferrin receptor, decreasing the receptor’s affmity for transferrin five to tenfold (24’1, and remainsassociatedwith the receptor throughout its intracellular journey [ZS’]. The predominant CysZ60+Tyr mutation (using the numbering in the mature protein without signal sequence)in hemochromatosislargely abolishesthis association[‘24-l, aswell asassociationwith and cell-surface expression 1261. &-microglobulin Functional significance of the associationof HFE with the transferrin receptor and its effect on receptor affinity for transferrin is still unclear, and the critical regulatory role of HFE in iron metabolism remains unexplained. HFE
Cellular
iron metabolism
Membrane transport of iron involves ferrireductases, ferroxidases and permeases,as highlighted in multifactorial yeast and bacterial systems (reviewed in [27’,28]). Mammalian cells display additional complexity because iron is typically acquired by receptor-mediated internalization of transferrin into acidic endosomalcompartments for releaseand membrane translocation, although nontransferrin-bound iron can alsobe taken up at the cell surface [29]. Generalized models must incorporate Fe(III)-binding sites, ferrireductases, ferroxidases and carriers mediating bilayer translocation of Fe(I1) [2’,3’,30-38,39’,40]. Advances in our understanding of mammalian iron uptake has led to the recent identification of transport molecules. Divalent
Physiological mechanisms of iron release from ferritin have been little explored. A persistent question is whether iron removal from ferritin is reversible or requires protein degradation.
g~yxqbdffn
of the protein [19]. A rofe for the transferrin receptor in modulating iron release from transfer&r in a p&dependent fashion has also been established [ZO]. In chimeric human/chicken receptors, the transferrin recognitinn site is localized to the exocytic carboxy-terminal region of the receptor (the chicken receptor does not accept human transferrin) [Zl], but the structure and precise location of the site are still unknown.
metal
transporter
1
A proton-coupled system that stimulates divalent metal uptake was detected in a functional expression cloning screen for Fe(I1) transporters in Xenopw oocytes [3’], thereby defining the function of a previously identified Nrampl
202
Bio-inorganic
chemistry
homolog [38]. Positional cloning further revealed that microcytic anemia mR mice have a mutation in the same gene [38]; an identical lesion was later found in the Belgrade b rat [39’]. DMTl is predicted to be a 561 amino acid protein which spans the membrane 12 times. Its activity couples uptake of Fe, Zn, Mn, Co, Cd, Cu, Ni and Pb with proton symport [3’] and its expression functionally complements yeast defects in Smfl and Smf2, homologs that are involved in divalent cation transport [41]. The mR mouse has defective dietary iron assimilation, suggesting that DMTl is an intestinal transporter [Z’]. In addition to impaired nontransferrin-bound iron transport, Belgrade b rat reticulocytes are also defective in transferrin-mediated uptake [42-44], suggesting that DMTl participates in iron’s exit from the endosome. Although it remains possible that these transport defects arise indirectly from deficits in other metals (e.g. Mn2+ deficiency [4.5]), exogenous DMTl expression does stimulate Fe(I1) uptake [3’,39’,46] and it localizes to early endosomes of the transferrin pathway, as well as to the plasma membrane [46]. The Fe(II)-stimulated current in DMTl-expressing oocytes is half-maximal at 2 PM Fe(I1) [3’]. Aithough the mk Gly185+Arg mutant is poorly expressed at the cell surface in transfection studies, this does not entirely account for the observed loss of transport activity, confirming the disruption of DMTl function in the mk mouse [46]. Stimulator
of Fe transport
Using an expressioncloning strategy similar to that outlined above, SFT was identified by screening for Fe(II1) uptake activity [47’]. SFT is a 338 amino acid protein with six membrane-spanning domains. An iron-binding motif (RExxE, where x can be any residue) comparable to functional elements of the yeast Ftrl permease[48] is found in the first intracellular loop; mutations at this site eliminate activity but do not interfere with surface expression [47’,48]. SFT migratesasa dimer of -85 kDa on reducing sodium dodecyl sulfate gels [47’], suggestingit is covalent assemblyinto a 12 membrane-spanning complex akin to DMTl [2’,3’,38]. Mechanistically, SFTstimulated uptake is energy-dependent [47’] but does not appear to be proton-coupled [49]. Unlike DMTl, SFT activity appearsrelatively selective for Fe; only Cd inhibits uptake [47’]. Moreover, SFT mediates Fe(II1) or Fe(I1) uptake [50], while DMTl only stimulates transport of Fe(I1) [3’]. SFT expressioncorrelateswith surface iron-binding sites with a dissociation constant (6 pM) closeto the apparent KM for SFTstimulated uptake (5 l&l) [50,51]. Increased maximum velocity values also correlate with SFT expressionlevels, implicating that it hasa direct role in transport [50], but because of the potential for endogenous transport modifiers, full verification of SFT (and DMTl) transport function in the absenceof additional factors awaits reconstitution studiesof purified protein. SFT is localized to endosomesand its expression stimulates iron assimilationfrom transferrin [47’,50.51]. Whether DMTl and SFT collaborate in endosomal transport is
unknown; although impaired, iron uptake persists in Belgrade reticulocytes, suggestingmultiple and redundant mechanismsdo exist [44]. A role for ferrireduction in endosomal transport has been implicated [52], and when cell-associated ferrireductase activity is impaired, SFT expressionstimulates uptake of Fe(I1) but not Fe(II1) [50]. Ferrireduction must alsobe a prerequisite for DMTl function [3’,39’,45], but cell-associatedactivity appears to be insufficient to support DMTl-mediated Fe(II1) uptake, suggestingthat SFT and DMTl differentially couple with ferrireductase(s). While spectrophotometric assayof ferricyanide reduction is straightforward and reliable, it should be noted that assayof ferrireductase activity using chromophoric heterocyclic Fe(I1) chelators may yield spurious results becausethe great affinity of the chelators for Fe(I1) can force reduction that would not take place in its absence. Ferrireductase
Transport-associated ferrireductases remain to be identified. Although studies indicate that mucosal transfer of iron is accelerated [53], increased iron reduction activity has also been reported for hemochromatosispatients [54]. Importantly, enhanced intestinal uptake of Fe(III), but not Fe(II), is displayed in animal models of hemochromatosis [55]. Thus, a ferrireductase, an Fe(I1) transporter and/or a complex mediating Fe(II1) uptake may be upregulated in this very common disease. Consistent with a role for DMTl, patients overload with other metals in addition to Fe [56]. Furthermore, SF?’ is upregulated in the liver, suggesting it contributes to the etiology of the disease [57]. Homeostasisis maintained by restricting iron transport, so important questions remain concerning the regulation of DMTl and SFT activities and how this balance is disrupted in hemochromatosis. An overview of cellular iron metabolism is presented in Figure 1.
Post-transcriptional control of iron homeostasis by iron regulatory proteins Iron homeostasisis post-transcriptionally regulated by the iron regulatory proteins (IRPs) 1 and 2 [S&59]. IRPs are cytosolic RNA-binding proteins that bind to a hairpin structure, known as the iron-responsive element (IRE). IREs are located in either the 5’ or 3’ untranslated regions (IJTRs) of specific mRNAs encoding proteins involved in iron and energy homeostasis.Structural studies showedthat the canonical IRE consists of a six-membered loop (CAGUGN), a variabie five basepair helix and an unpaired bulging C or CGU loated six bases 5’ to the first nucleotide in the loop [60,61]. IREs were first identified in the ferritin 5’ IJTR, and later in the 5’ UTRs of the Krebs cycle enzymes mitochondrial (m)-aconitase and Drosophila meLangonaster succinate dehydrogenase, as well as in the heme biosynthetic enzyme erythroid aminolevulinate synthase. Binding of IRPs to the ferritin 5’ IRE represses translation by preventing recruitment of the 40s small ribosomal subunit to the mRNA [62]. Five IREs have been identified in the 3’ UTR of the transferrin receptor mRNA and one in the 3’ UTR of DMTl tnRNA [3-j. The binding
Iron metabolism
Figure
Aisen,
Wessling-Resnick
and
Leibold
203
1
Fe(H)
e
Fe(lll)
Diferric
Tf
Fe(lll)/Fe(ll) I
I
m-face
I
endosome
Fe-proteins
.
IRPs
Ferritin Current
of IRPs to the transferrin receptor 3’ IREs mRNA from endonucleolytic cleavage [63]. Mechanisms
of iron regulatory
protein
Cells acquire iron by the internalization of transferrin-Fe(lll) (Tf) with the transferrin receptor (TfR). Transferrin-independent transport by DMTl and SFT also can be detected. The low endosomal pH results in the release of Feflll) from the Tf-TfR complex. Reduction of iron is required for the endosomal transport of iron; however, transport-associated ferrireductases have not been identified. Iron enters an intermediate pool that is sensed by IRPl and IRP2. Iron regulation of IRPs results in increased ferritin translation, leading to iron sequestration, and destablization of TfR mRNA.
protects this
1 action
A clue to the mechanism of IRPl regulation by iron was revealed when IRPl was shown to be the cytosolic counterpart of m-aconitase. Aconitase is a [4Fe--4S]-containing Krebs cycle enzyme that catalyzes the isomerization of citrate to isocitrate. Although IRPl and m-aconitaseare only 30% identical, IRPl contains all the aconitase active sites and a [4FellS] cluster. Iron regulates the RNA-binding activity of IRPl: in iron-depleted cells, IRPl binds IREs with high affinity, whereas in iron-repleted cells, iron converts apo-IRPl into the active [4Fe-4S] aconitase, nonRNA-binding form. RNA binding and aconitaseactivities are mutually exclusive, and the switch between these activities occurs without changesin IRPl protein levels. IRP’2 sharesabout 62% identity with IRPl, but despite sequencesimilarities, it is thought not to form a [4Fe--4S] cluster, and consequently lacks aconitase activity. (For a recent review on these Fe-S clusters,seeBeinert and Kiley’s review in this issue[pp 1X-157].) IRPZ is regulated by proteolysis by the proteasome[64,65], which is mediated by an iron-dependent oxidation mechanismrequiring a unique 73 amino acid domaincontaining three cysteine residues[6.5,66]. The significanceof two IRPs in iron homeostasisis unclear. One possibility is that they bind specific IREs in viva, which is consistentwith studiesshowing that IRPl and IRPZ bind to specific IREs in aitm [67-70.1.Differences in the binding
Opinion
in Chemical
Biology
affmities of IRPl and IRPZ and in the concentration of IRPl and IRPZ in cells, coupled with the differential regulation by oxidants and the nitric oxide radical (NO’), may fine-tune IRE mRNA responsesto diverse signals. Effects of oxidative
stress
In addition to iron, IRPl is also activated by NO’, H,O, [58,59,71] and during hypoxia-reoxygenation [72], and is accompanied by the inactivation of aconitase activity. The mechanism regulating H202-induced IRPl activation differs from NO’. Electron paramagnetic resonance studies have shown that the inactivation of aconitase by NO’ is caused by the disassemblyof the [4FeAS] cluster to an [3Fe--4S] cluster [73]. H,O, activation is dependent on extracellular signaling events [74,75], suggestingthat cluster disassembly alone cannot fully account for H202-induced IRPl activation and that signaling pathways are involved. IRPl and IRPZ RNA-binding activity are induced by protein kinase C phosphorylation [76], which is consistent with the notion that a stress-signalingpathways may be involved. Conflicting resultsconcerning the effects of NO’ on IRPZ have been reported. IRPZ regulation may be more complex than thought, since signalssuch asphosphorylation [76], cysteine oxidation [66] and growth, in addition to iron, may modulate IRPZ activity. Hypoxia reoxygenation activation of IRPl showssimilarities with H,O, in that reactive oxygen speciesmay be involved in both signalingand cluster oxidation [72]. Hypoxia resulted in IRPl inactivation [77] and was accompanied by an
204
Bio-inorganic
chemistry
increase in aconitase activity and the activation of IRPZ [72]. The decrease in IRPl activity during hypoxia appears to be due to the stabilization of cytoplasmic aconitase [4Fe-4S], whereas the activation of IRPZ is caused by an increase in IRPZ stability (EA Leibold, unpublished data). In contrast to cell models of oxidative stress, post-ischemic reperfusion injury in rat liver resulted in IRPl and IRPZ inactivation and increased ferritin synthesis [78]. The discrepancy between these studies is unclear, but may be related to differences between in viva and in v,itro systems.
Conclusions A diversity of metabolic pathways has evolved to accomadate organisms’ need for iron, a metal that is essential to life but is toxic in excess, and that therefore exposes species from bacteria to man to the dual hazards of overload and deficiency. Principal features of some of these pathways in mammalian cells, involving transferrin, the transferrin receptor and ferritin, have been known for years, but the fine details necessary to understand their functions in iron transport and storage at the molecular level are still to be fully revealed. New proteins involved in iron transport have been discovered, notably DMTl and SFT, but again their precise functions and (especially) their interactions are unclear. The homeostatic regulatory events in iron metabolism are rich and complex subjects for investigation. What was unforeseeable a few years ago is now common knowledge, and what will be understood in the coming years probably eludes speculation now.
Adrnowledgments \Ve offer our thanks ro colleagues who shared information and an apology for insufficient space co credit each of a very contributions co this field.
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of of the
Iron metabolism Philip Aisen *, Marianne The
understanding
of iron metabolism
has been enormously expanded findings about the functioning receptor discovery previously
proteins
at the molecular
in recent of transferrin,
and ferritin. Other recent of the hemochromatosis unknown
Wessling-Resnickt
developments include gene HFE, identification
involved
in iron transport,
metal transporter expanded insights
1 and stimulator of Fe transport, and into the regulation and expression of
proteins
involved
in iron metabolism.
principal although
participants in iron transport have the complexity of such interactions
understood. concepts biology continue
Interactions
Correlated efforts involving of crystallography, spectroscopy applied
to cellular
to be, particularly
processes
level
years by new the transferrin the of divalent
among
been uncovered, is still incompletely
techniques and and molecular have
been,
and should
Opinion
in Chemical
Biology
1999,
3:200-206
http://biomednet.com/elecref/1367593100300200 0 Elsevier
Science
Ltd ISSN
and cloned [2’,3’]. Another protein functioning asa stimulator of iron transport, stimulator of Fe transport (SFT), has been identified and cloned [4]. New insights into the function, regulation and interactions of major proteins of iron metabolism, including transferrin, the transferrin receptor and ferritin, have similarly expanded. Nevertheless, fine details of molecular mechanismsinvolved in iron transport and storageoften remain elusive. In this brief review, we will focus on recent developments in the broad area of iron metabolism-proteins of iron metabolism,cellular transport of iron, and the regulation of iron metabolism- emphasizing uncertainties to be resolved.
Proteins
of iron metabolism
Transferrin
revealing.
Addresses *Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; e-mail: [email protected] Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115-6021, USA; e-mail: [email protected] IEccles Program in Human Molecular Biology and Genetics and the Department of Medicine, University of Utah, Building 533, Room 4220, Salt Lake City, UT 84112, USA; e-mail: [email protected] Current
and Elizabeth A Leiboldl
Transferrin, the iron-transporting plasma protein, provides most of the iron for the physiological needs of iron-requiring cells, and is normally the only known source of iron for hemoglobin synthesis. The detailed structure of the serum transferrin molecule, revealed by X-ray crystallography, shows it to be, as predicted from internal homology, a bilobal protein with each lobe (designated the N and C lobes for their respective sequences in the transferrin molecule) bearing a single metal-binding site and comprising two domains [5,6]. The iron ligands of all known transferrins are two tyrosine residues,a histidine, aspartate and a synergistic carbonate anion linked to both metal and protein. Without such a linking anion, the iron-binding function of transferrin is lost.
1367-5931
Abbreviations DMTl divalent metal transporter H heavy/heart HFE hemochromatosis gene IRE iron-responsive element iron regulatory protein IRP L light/liver m mitochondrial nitric oxide radical NO stimulator of Fe transport SFT UTR untranslated region
1
Introduction In the past few years, our understandingof iron metabolism in unicellular and multicellular organisms ranging from microbes to man has advanced dramatically. HFE, the long sought-after gene responsiblefor hereditary hemochromatosis, an iron-overloading disorder that, untreated, leads to disability and death, hasbeen identified and cloned [ 11,and interactions of the HFE protein with other participants in iron metabolism is under active study. A protein implicated to be a principal iron transporter in intestinal mucosalcells and to alsobe involved in intracellular transmembranetransport, divalent metal transporter 1 (DMTl) (formerly natural resistance-associatedmacrophage protein 2 [NrampZ] or divalent cation transporter 1 [DCTl]), has been identified
In addition to the role of these iron ligands in iron binding and release, the protein fabric itself strongly regulates release. Iron release is generally more facile from the N lobe, particularly at the low pH (near 5.6) of the endosome,where iron is releasedwithin the cell. Protonation of two lysine residues, neighbors in space but situated on opposite domains, results in electrostatic repulsion of the domains as a dominant event in iron release from the N lobe [7,8] but not in the C lobe, which lacks the dilysine pair. A variety of site-directed mutagenesisstudies indicate that residuesnot directly involved in iron binding alsofunction in modulating iron release,and that such outer-shell or remote functions may differ in the two lobes [9’,10’]. Mechanisms of iron binding have received relatively little attention. Most studies suggest that binding of the synergistic anion precedes iron binding [ 111,and that a seriesof deprotonation reactions results in a closing of the two domains of the binding lobe to lock iron into place [12]. A protein with remarkable similarity to transferrin in its iron-binding properties, but without sequence homology to transferrin, has been isolated from the periplasmic space of pathogenic ZV&~~etiia [13’]. Thought to act as an iron
Iron metabolism
;axxeppx fKm hiw L Llalls -- - F-ddl, --* XiEs3SjX3Skiig wx~ss the pei$asmic space to rhe cyK3ph~m
ihC
S-Si>X
af the
Aisen,
Wessling-Resnick
and Leibold
is a homndimetic
g3ycqmkm,
201
&ii
infec-
The
rransferfm
receptor
k3
tious organism, the protein resembles transferrin in displaying two ryrosine ljgands lbur oifiesent>y disposed from the arrangement in transferrin) and one histidine lig-
two subunits linked by a pair of disulfide bonds at or near the inrramembranous porrjon of the mo)ecuk. Of she four glycosylation sites, three are N-linked and one is O-linked;
and, but wirh ghtamak in place of aspar~ate. A partic&dy intriguing finding is the presenceof a noncovalently bound
N-linked &king
phosphate group, apparently acting as the synergistic anion. Whether the bacterial protein will also accommodate carbonate remains to be determined. Ferritin
Ferritin is involved in iron storage and detoxification in microbial, plant and animal species.The mammalianferritin molecule is a heteropolymer of 24 subunits of two types, designated H (for heart or heavy) and L (for liver or light), depending on the organs in which they predominate and their relative molecular weights (typically 19,000-21,000). Sequence identity between H and I, chains is >SO%;the ratio of H to L subunits is a function of the tissue of origin. Subunits can spontaneouslyassembleinto a hollow spherical protein, the interior of which accommodates4,000 or more iron atoms asferrihydrite, FeOOH, although most isolated ferritins contain closer to 2,000 iron atoms. Four hydrophilic channelsof threefold symmetry, and three fourfold hydrophobic channelspierce the protein shell. A widely accepted but still disputed model identifies sites present only within H chains where a combination of glutamate, tyrosine and histidine residues form a ferroxidase center that functions to bind Fe(I1) dimers and catalyze their oxidation via a peroxodiferric intermediate (that was observed in freeze-quench Mossbauer spectroscopy) [14]. Fe(II1) is then conveyed through undefined intrasubunit routes and the hydrophilic channels to the protein cavity, where core formation initiates at carboxyl groups presented by L chain glutamate residues [15] to complete the three-stage process of Fe(II) binding, oxidation and core formation. Iron uptake appears to be driven by electrostatic gradients [16] and facilitated by plasticity in protein conformation, creating pathways (not evident in the crystal structure) for iron to follow. Once core formation is underway the growing core serves to promote iron oxidation, with a 1:4 stoichiometry of dioxygen consumed to iron oxidized, rather than 1~2,as seen in ferroxidase site oxidation [17’]. The alternative view is that Fe(I1) oxidation is carried out not by ferritin but by ceruloplasmin, a multicopper-containing plasmaprotein with known ferroxidase activity, or an intracellular analog of ceruloplasmin [ 181.The resulting Fe(II1) is then transported to the ferritin cavity for mineralization into the core. The controversy continues.
has a cfi&d
The hemochromatosis
de
in fdbing
and craf-
gene
and its product
has been identified, cloned and expressed, the missense mutation responsible for most cases of hemochromatosisfound and the structure of its product determined by X-ray crystallography [1,22”]. The HFE protein is a 343-residue transmembrane glycoprotein homologous to class I major histocompatibility complex (MHC) proteins. Like other MHC proteins, it associates with &-microglobulin at the cell surface, with disruption of such associationleading to generalized iron overload [23]. HFE also associateswith the transferrin receptor, decreasing the receptor’s affmity for transferrin five to tenfold (24’1, and remainsassociatedwith the receptor throughout its intracellular journey [ZS’]. The predominant CysZ60+Tyr mutation (using the numbering in the mature protein without signal sequence)in hemochromatosislargely abolishesthis association[‘24-l, aswell asassociationwith and cell-surface expression 1261. &-microglobulin Functional significance of the associationof HFE with the transferrin receptor and its effect on receptor affinity for transferrin is still unclear, and the critical regulatory role of HFE in iron metabolism remains unexplained. HFE
Cellular
iron metabolism
Membrane transport of iron involves ferrireductases, ferroxidases and permeases,as highlighted in multifactorial yeast and bacterial systems (reviewed in [27’,28]). Mammalian cells display additional complexity because iron is typically acquired by receptor-mediated internalization of transferrin into acidic endosomalcompartments for releaseand membrane translocation, although nontransferrin-bound iron can alsobe taken up at the cell surface [29]. Generalized models must incorporate Fe(III)-binding sites, ferrireductases, ferroxidases and carriers mediating bilayer translocation of Fe(I1) [2’,3’,30-38,39’,40]. Advances in our understanding of mammalian iron uptake has led to the recent identification of transport molecules. Divalent
Physiological mechanisms of iron release from ferritin have been little explored. A persistent question is whether iron removal from ferritin is reversible or requires protein degradation.
g~yxqbdffn
of the protein [19]. A rofe for the transferrin receptor in modulating iron release from transfer&r in a p&dependent fashion has also been established [ZO]. In chimeric human/chicken receptors, the transferrin recognitinn site is localized to the exocytic carboxy-terminal region of the receptor (the chicken receptor does not accept human transferrin) [Zl], but the structure and precise location of the site are still unknown.
metal
transporter
1
A proton-coupled system that stimulates divalent metal uptake was detected in a functional expression cloning screen for Fe(I1) transporters in Xenopw oocytes [3’], thereby defining the function of a previously identified Nrampl
202
Bio-inorganic
chemistry
homolog [38]. Positional cloning further revealed that microcytic anemia mR mice have a mutation in the same gene [38]; an identical lesion was later found in the Belgrade b rat [39’]. DMTl is predicted to be a 561 amino acid protein which spans the membrane 12 times. Its activity couples uptake of Fe, Zn, Mn, Co, Cd, Cu, Ni and Pb with proton symport [3’] and its expression functionally complements yeast defects in Smfl and Smf2, homologs that are involved in divalent cation transport [41]. The mR mouse has defective dietary iron assimilation, suggesting that DMTl is an intestinal transporter [Z’]. In addition to impaired nontransferrin-bound iron transport, Belgrade b rat reticulocytes are also defective in transferrin-mediated uptake [42-44], suggesting that DMTl participates in iron’s exit from the endosome. Although it remains possible that these transport defects arise indirectly from deficits in other metals (e.g. Mn2+ deficiency [4.5]), exogenous DMTl expression does stimulate Fe(I1) uptake [3’,39’,46] and it localizes to early endosomes of the transferrin pathway, as well as to the plasma membrane [46]. The Fe(II)-stimulated current in DMTl-expressing oocytes is half-maximal at 2 PM Fe(I1) [3’]. Aithough the mk Gly185+Arg mutant is poorly expressed at the cell surface in transfection studies, this does not entirely account for the observed loss of transport activity, confirming the disruption of DMTl function in the mk mouse [46]. Stimulator
of Fe transport
Using an expressioncloning strategy similar to that outlined above, SFT was identified by screening for Fe(II1) uptake activity [47’]. SFT is a 338 amino acid protein with six membrane-spanning domains. An iron-binding motif (RExxE, where x can be any residue) comparable to functional elements of the yeast Ftrl permease[48] is found in the first intracellular loop; mutations at this site eliminate activity but do not interfere with surface expression [47’,48]. SFT migratesasa dimer of -85 kDa on reducing sodium dodecyl sulfate gels [47’], suggestingit is covalent assemblyinto a 12 membrane-spanning complex akin to DMTl [2’,3’,38]. Mechanistically, SFTstimulated uptake is energy-dependent [47’] but does not appear to be proton-coupled [49]. Unlike DMTl, SFT activity appearsrelatively selective for Fe; only Cd inhibits uptake [47’]. Moreover, SFT mediates Fe(II1) or Fe(I1) uptake [50], while DMTl only stimulates transport of Fe(I1) [3’]. SFT expressioncorrelateswith surface iron-binding sites with a dissociation constant (6 pM) closeto the apparent KM for SFTstimulated uptake (5 l&l) [50,51]. Increased maximum velocity values also correlate with SFT expressionlevels, implicating that it hasa direct role in transport [50], but because of the potential for endogenous transport modifiers, full verification of SFT (and DMTl) transport function in the absenceof additional factors awaits reconstitution studiesof purified protein. SFT is localized to endosomesand its expression stimulates iron assimilationfrom transferrin [47’,50.51]. Whether DMTl and SFT collaborate in endosomal transport is
unknown; although impaired, iron uptake persists in Belgrade reticulocytes, suggestingmultiple and redundant mechanismsdo exist [44]. A role for ferrireduction in endosomal transport has been implicated [52], and when cell-associated ferrireductase activity is impaired, SFT expressionstimulates uptake of Fe(I1) but not Fe(II1) [50]. Ferrireduction must alsobe a prerequisite for DMTl function [3’,39’,45], but cell-associatedactivity appears to be insufficient to support DMTl-mediated Fe(II1) uptake, suggestingthat SFT and DMTl differentially couple with ferrireductase(s). While spectrophotometric assayof ferricyanide reduction is straightforward and reliable, it should be noted that assayof ferrireductase activity using chromophoric heterocyclic Fe(I1) chelators may yield spurious results becausethe great affinity of the chelators for Fe(I1) can force reduction that would not take place in its absence. Ferrireductase
Transport-associated ferrireductases remain to be identified. Although studies indicate that mucosal transfer of iron is accelerated [53], increased iron reduction activity has also been reported for hemochromatosispatients [54]. Importantly, enhanced intestinal uptake of Fe(III), but not Fe(II), is displayed in animal models of hemochromatosis [55]. Thus, a ferrireductase, an Fe(I1) transporter and/or a complex mediating Fe(II1) uptake may be upregulated in this very common disease. Consistent with a role for DMTl, patients overload with other metals in addition to Fe [56]. Furthermore, SF?’ is upregulated in the liver, suggesting it contributes to the etiology of the disease [57]. Homeostasisis maintained by restricting iron transport, so important questions remain concerning the regulation of DMTl and SFT activities and how this balance is disrupted in hemochromatosis. An overview of cellular iron metabolism is presented in Figure 1.
Post-transcriptional control of iron homeostasis by iron regulatory proteins Iron homeostasisis post-transcriptionally regulated by the iron regulatory proteins (IRPs) 1 and 2 [S&59]. IRPs are cytosolic RNA-binding proteins that bind to a hairpin structure, known as the iron-responsive element (IRE). IREs are located in either the 5’ or 3’ untranslated regions (IJTRs) of specific mRNAs encoding proteins involved in iron and energy homeostasis.Structural studies showedthat the canonical IRE consists of a six-membered loop (CAGUGN), a variabie five basepair helix and an unpaired bulging C or CGU loated six bases 5’ to the first nucleotide in the loop [60,61]. IREs were first identified in the ferritin 5’ IJTR, and later in the 5’ UTRs of the Krebs cycle enzymes mitochondrial (m)-aconitase and Drosophila meLangonaster succinate dehydrogenase, as well as in the heme biosynthetic enzyme erythroid aminolevulinate synthase. Binding of IRPs to the ferritin 5’ IRE represses translation by preventing recruitment of the 40s small ribosomal subunit to the mRNA [62]. Five IREs have been identified in the 3’ UTR of the transferrin receptor mRNA and one in the 3’ UTR of DMTl tnRNA [3-j. The binding
Iron metabolism
Figure
Aisen,
Wessling-Resnick
and
Leibold
203
1
Fe(H)
e
Fe(lll)
Diferric
Tf
Fe(lll)/Fe(ll) I
I
m-face
I
endosome
Fe-proteins
.
IRPs
Ferritin Current
of IRPs to the transferrin receptor 3’ IREs mRNA from endonucleolytic cleavage [63]. Mechanisms
of iron regulatory
protein
Cells acquire iron by the internalization of transferrin-Fe(lll) (Tf) with the transferrin receptor (TfR). Transferrin-independent transport by DMTl and SFT also can be detected. The low endosomal pH results in the release of Feflll) from the Tf-TfR complex. Reduction of iron is required for the endosomal transport of iron; however, transport-associated ferrireductases have not been identified. Iron enters an intermediate pool that is sensed by IRPl and IRP2. Iron regulation of IRPs results in increased ferritin translation, leading to iron sequestration, and destablization of TfR mRNA.
protects this
1 action
A clue to the mechanism of IRPl regulation by iron was revealed when IRPl was shown to be the cytosolic counterpart of m-aconitase. Aconitase is a [4Fe--4S]-containing Krebs cycle enzyme that catalyzes the isomerization of citrate to isocitrate. Although IRPl and m-aconitaseare only 30% identical, IRPl contains all the aconitase active sites and a [4FellS] cluster. Iron regulates the RNA-binding activity of IRPl: in iron-depleted cells, IRPl binds IREs with high affinity, whereas in iron-repleted cells, iron converts apo-IRPl into the active [4Fe-4S] aconitase, nonRNA-binding form. RNA binding and aconitaseactivities are mutually exclusive, and the switch between these activities occurs without changesin IRPl protein levels. IRP’2 sharesabout 62% identity with IRPl, but despite sequencesimilarities, it is thought not to form a [4Fe--4S] cluster, and consequently lacks aconitase activity. (For a recent review on these Fe-S clusters,seeBeinert and Kiley’s review in this issue[pp 1X-157].) IRPZ is regulated by proteolysis by the proteasome[64,65], which is mediated by an iron-dependent oxidation mechanismrequiring a unique 73 amino acid domaincontaining three cysteine residues[6.5,66]. The significanceof two IRPs in iron homeostasisis unclear. One possibility is that they bind specific IREs in viva, which is consistentwith studiesshowing that IRPl and IRPZ bind to specific IREs in aitm [67-70.1.Differences in the binding
Opinion
in Chemical
Biology
affmities of IRPl and IRPZ and in the concentration of IRPl and IRPZ in cells, coupled with the differential regulation by oxidants and the nitric oxide radical (NO’), may fine-tune IRE mRNA responsesto diverse signals. Effects of oxidative
stress
In addition to iron, IRPl is also activated by NO’, H,O, [58,59,71] and during hypoxia-reoxygenation [72], and is accompanied by the inactivation of aconitase activity. The mechanism regulating H202-induced IRPl activation differs from NO’. Electron paramagnetic resonance studies have shown that the inactivation of aconitase by NO’ is caused by the disassemblyof the [4FeAS] cluster to an [3Fe--4S] cluster [73]. H,O, activation is dependent on extracellular signaling events [74,75], suggestingthat cluster disassembly alone cannot fully account for H202-induced IRPl activation and that signaling pathways are involved. IRPl and IRPZ RNA-binding activity are induced by protein kinase C phosphorylation [76], which is consistent with the notion that a stress-signalingpathways may be involved. Conflicting resultsconcerning the effects of NO’ on IRPZ have been reported. IRPZ regulation may be more complex than thought, since signalssuch asphosphorylation [76], cysteine oxidation [66] and growth, in addition to iron, may modulate IRPZ activity. Hypoxia reoxygenation activation of IRPl showssimilarities with H,O, in that reactive oxygen speciesmay be involved in both signalingand cluster oxidation [72]. Hypoxia resulted in IRPl inactivation [77] and was accompanied by an
204
Bio-inorganic
chemistry
increase in aconitase activity and the activation of IRPZ [72]. The decrease in IRPl activity during hypoxia appears to be due to the stabilization of cytoplasmic aconitase [4Fe-4S], whereas the activation of IRPZ is caused by an increase in IRPZ stability (EA Leibold, unpublished data). In contrast to cell models of oxidative stress, post-ischemic reperfusion injury in rat liver resulted in IRPl and IRPZ inactivation and increased ferritin synthesis [78]. The discrepancy between these studies is unclear, but may be related to differences between in viva and in v,itro systems.
Conclusions A diversity of metabolic pathways has evolved to accomadate organisms’ need for iron, a metal that is essential to life but is toxic in excess, and that therefore exposes species from bacteria to man to the dual hazards of overload and deficiency. Principal features of some of these pathways in mammalian cells, involving transferrin, the transferrin receptor and ferritin, have been known for years, but the fine details necessary to understand their functions in iron transport and storage at the molecular level are still to be fully revealed. New proteins involved in iron transport have been discovered, notably DMTl and SFT, but again their precise functions and (especially) their interactions are unclear. The homeostatic regulatory events in iron metabolism are rich and complex subjects for investigation. What was unforeseeable a few years ago is now common knowledge, and what will be understood in the coming years probably eludes speculation now.
Adrnowledgments \Ve offer our thanks ro colleagues who shared information and an apology for insufficient space co credit each of a very contributions co this field.
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