Blood Iron Homeostasis: Newly Discovered Proteins and
Iron Imbalance
Mark R. Bleackley1, Ann Y.K. Wong1, David M. Hudson, Christopher H-Y. Wu, and Ross T.A. MacGillivray
In biological systems, iron exerts 2 contrasting effects.
The chemical reactivity of iron is essential for the
biological activities of proteins such as hemoglobin,
ribonucleotide reductase, the cytochromes, and aconi-
tases. However, free iron in a cell has the propensity to
generate free radicals which can damage cellular
components containing proteins, lipids, and nucleic
acids. To maintain the balance between iron as an
essential nutrient and iron as a potential cytotoxin, a
number of biological protective mechanisms have
ORE THAN HALF of the 3 to 4 g of iron in
M a well-nourished human is bound to hemo-
globin in red blood cells (RBCs) and their
precursors. Each day, 20 to 25 mg of iron is
required to support the synthesis of hemoglobin in
reticulocytes. The chemical properties of iron that
make it such a beneficial element also lead to a
propensity of free iron and the subsequent forma-
tion of damaging free radicals via the Fenton series
of reactions. As a result, organisms have evolved
complex biochemical mechanisms for maintaining
a balance between iron as an essential element and
iron as a cytotoxic agent. With the exception of a
few members of the genus Lactobacillus and some
strains of Bacillus, iron is essential for the viability
of all organisms.1 Two major redox states exist for
iron (ferrous or Fe(II) and ferric or Fe(III)), but both
higher and lower valences have been observed in
the laboratory. Under physiologic conditions, iron
exists in a number of biochemically available redox
states that allow it to participate in single electron
transfer reactions. Binding of different ligands to
iron can vary the potential of the Fe(II)/Fe(III)
redox couple allowing iron to meet the require-
ments of a wide range of cellular reactions. Iron in
proteins is often found complexed in heme (such as
hemoglobin) or in iron-sulfur (Fe-S) clusters (such
as aconitases). Other proteins (nonheme iron
proteins including ribonucleotide reductase and
transferrin [Tf]) bind iron in a variety of different
ways.
During the past few years, several molecular
genetic approaches have revealed the functions of
many new iron-related proteins. In this review, we
discuss the strategies used to identify proteins such
as hephaestin (Hp), ferroportin, divalent metal
Transfusion Medicine Reviews, Vol 23, No 2 (April), 2009: pp 103-123
evolved. As shown in the thalassemias, iron imbalance
can have devastating effects on human health. Recently,
several new proteins have been described that play
critical roles in iron regulation including the master
regulator of iron metabolism (hepcidin). In this review,
we discuss the new knowledge that has arisen from
studies in yeast and in humans, and we show how these
studies are shedding new light on some well-known
human disorders.
© 2009 Elsevier Inc. All rights reserved.
transporter 1, duodenal cytochrome b, hemojuvelin,
and hepcidin. We then discuss the proposed
functions of these proteins in iron homeostasis
while identifying gaps in our current knowledge.
Abbreviations: Aα, alpha-amyloid peptide; APP, amyloid β
precursor protein; β2M, beta 2 microglobulin; BMP, bone
morphogenetic protein; Cp, ceruloplasmin; cytb561, cytochrome
b561; DCT1, divalent cation transporter; Dcytb, duodenal
cytochrome b; DMT1, divalent metal transporter 1; FPN1,
ferroportin-1; FRDA, Friedreich's ataxia; FXN, frataxin; GPI,
glycosylphosphatidylinositol; HCP1, heme carrier protein 1; HFE,
hemochromatosis protein; HH, hereditary hemochromatosis;
HIF1α, hypoxia inducible factor-1 α; HJV, hemojuvelin; Hp,
hephaestin; IMP, integrin-mobilferrin pathway; IRE, iron response
element; IRP, iron response protein; Lf, lactoferrin; LIP, labile iron
pool; mHJV, glycosylphosphatidylinositol-anchored hemojuvelin;
MTf, melanotransferrin; PCFT, proton-coupled folate transporter;
RBCs, red blood cells; ROS, reactive oxygen species; sHJV, soluble
hemojuvelin; SLA, sex-linked anemia; sMTf, soluble
melanotransferrin; Tf, transferrin; TfR, transferrin receptor; TM,
transmembrane; UTR, untranslated region.
From the Centre for Blood Research and Department of
Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, BC, Canada.
This study was supported in part by a grant from the
Canadian Blood Services–Canadian Institutes of Health
Research (CIHR) Program in Blood Utilization and
Conservation awarded to RTAM. MRB was supported by a
graduate studentship from the Strategic Training Program in
Transfusion Science (funded by the CIHR and the Heart and
Stroke Foundation of Canada). AYKW was supported by a
graduate studentship from CBS.
1
These authors contributed equally to this work.
Address reprint requests to Ross T.A. MacGillivray, Centre for
Blood Research, University of British Columbia, Vancouver, BC,
Canada V6T 1Z3. E-mail: macg@interchange.ubc.ca
0887-7963/08/$ - see front matter
© 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.tmrv.2008.12.001
103
104
Finally, we discuss the disorders that result from a
deficiency of these new proteins including effects
within iron regulation as well as some newly
recognized neurodegenerative diseases.
CHEMISTRY AND BIOCHEMISTRY OF IRON
Located in the middle of the periodic table, iron
is a transition metal that exists in oxidation states
ranging from −II, as in the Fe(CO)42− anion, to +VI,
as in the ferrate ion FeO42−.2 Biochemically, Fe(II)
and Fe(III) are the most relevant oxidation states.
Transfer of a single electron between these 2 states
is readily accomplished with ascorbate or molecular
oxygen. Fe(IV) and Fe(V) are encountered in
biology but only as reactive intermediates in the
catalytic mechanisms of some iron-containing
enzymatic processes.3 The potential of the Fe(III)/
Fe(II) redox couple (0.77 V)2 can vary by up to 1 V
in response to different environments,4,5 particu-
larly when influenced by protein-bound coordina-
tion ligands. This variability in redox potential
contributes significantly to the role of iron as an
essential biological metal. Within the range of
biological oxidants and reductants (+0.82V to
−0.32V, molecular oxygen and pyridine nucleo-
tides, respectively), electron transfers facilitated by
iron are reversible.6
PREVALENCE OF IRON IN BIOLOGICAL
SYSTEMS
On examination of the history of the earth and
evolution of life, it is clear that abundance,
availability, and suitability of iron have been critical
to life. In the earth's crust, iron is the fourth most
abundant element and second most abundant metal.
Stars, such as our sun, produce energy through the
formation of helium via nuclear fusion of hydro-
gen.7 Helium builds up in the core of the star
leading to the formation of a red giant. As the
temperature increases to 108 K, helium fuses to
form large quantities of carbon, and oxygen
becomes the major energy source.8 In larger stars,
this process occurs more rapidly (10 million years).
The additional heat from the fusion reactions (109
K) provides the energy7 to fuse nuclei up to the
mass of iron but is insufficient for fusions forming
heavier elements. As a result, large amounts of iron
accumulate in the core. Our solar system was
formed via aggregation of material from an earlier
star that exploded. Thus, these cosmochemical
BLEACKLEY ET AL
phenomena explain why there is so much iron on
and within the earth.2
When life first evolved on earth, the atmosphere
is believed to have been anoxic. Under these
conditions, Fe(II) was stable (as it did not auto-
oxidize) and the concentration of dissolved ferrous
iron in the oceans was around 50 μmol/L. The
abundance of iron in the oceans, combined with
chemical properties of iron, made it well suited for
biological systems. In the absence of oxygen,
copper (which is also capable of single electron
transfer reactions) existed predominantly in the
insoluble and thus nonbioavailable Cu(I) state.
Early life exploited Fe(II) chemistry and avail-
ability to carry out chemical reactions necessary for
survival. Evolution of photosynthetic bacteria 109
years ago began the shift to the aerobic atmosphere
found today. However, it is estimated that it took
200 to 300 million years to oxidize all of the iron in
the oceans with the resulting precipitation and
sedimentation of the Fe(III). Today, evidence of the
shift to an aerobic atmosphere can be seen as red
bands in sedimentary rock. In contrast, the oxida-
tion of copper to Cu(II) increased solubility and
thus bioavailability allowing for the evolution of
copper-catalyzed biological reactions. Bioavailable
Fe(II) was no longer prevalent, leading to a mass
extinction of many life forms. Surviving species
had evolved mechanisms for scavenging and
solubilizing Fe(III) and reducing it to Fe(II).
However, Fe(II) can react with environmental
oxygen, causing production of hydroxyl radicals
through the Haber-Weiss-Fenton reaction sequence.
Extracellular matrices of unicellular organisms
were subject to destruction by these damaging
radicals. In turn, organisms evolved matrices of
crosslinked connective tissue using free radical
polymerization. This is thought to correspond with
the beginning of multicellular life.1
IRON METABOLISM IN YEAST
Baker's yeast (Saccharomyces cerevisiae) has
proved to be a valuable model organism in the
study of iron metabolism. A high degree of
conservation exists between the cellular components
of iron metabolism in humans and S cerevisiae (see
Table 1). As shown in Figure 1, there are 3 iron
uptake pathways in S cerevisiae: 2 low affinity iron
pathways (Fet4 and Smf1) and one high affinity
pathway (Fet3/Ftr1). There is also a siderophore
scavenging pathway that is not shown in Figure 1.
IRON IMBALANCE AND DISEASE 105

Table 1. Proteins of Human Iron Metabolism and Homologs in S cerevisiae

Human Gene/
Protein Function in Humans Yeast Homolog Function in Yeast

Dcytb Membrane ferric reductase None but yeast contains Not applicable
nonhomologous ferric
reductases such as Fre1/2
DMT1 Dietary iron absorption at the SMF1/2 Broad specificity divalent and
apical membrane of duodenal trivalent metal transporter
enterocytes, release of iron
from transferrin cycle
endosomes
Ferritin Multimeric iron storage protein None Not applicable
Ferroportin-1 Exports iron out of duodenal None Not applicable
enterocytes, macrophages,
and a variety of neural cells
Frataxin Regulates mitochondrial iron Yfh1 Regulates mitochondrial iron
accumulation, thought to accumulation, thought to
promote Fe(II) availability, promote Fe(II) availability,
involved in maturation of heme involved in maturation of heme
and Fe-S cluster proteins and Fe-S cluster proteins
Human ATX1 homologue Cytoplasmic Cu Atx1 Cytoplasmic Cu
metallochaperone metallochaperone that
transports copper to Ccc2
Hephaestin Multicopper ferroxidase Fet3 Multicopper ferroxidase
involved in iron export from required for high affinity
duodenal enterocytes iron uptake
Heme carrier protein 1 Apical membrane heme None Not applicable
transporter
Mitoferrin Mitochondrial membrane iron Mrs3/4 Mitochondrial membrane iron
transporter transporter
Transferrin Binding of Fe3+ in blood None Not applicable
Transferrin receptor 1 Receptor for Tf-Fe, essential for None Not applicable
iron uptake from blood
Wilson/Menkes protein Golgi Cu transporters Ccc2 Cu transporting P-type ATPase,
responsible for Cu uptake into
the late Golgi for incorporation
into Fet3

Once inside the cell, iron is trafficked to mitochondria
where it is transported across the membrane by
proteins such as Mrs3/4. Inside the mitochondrion,
Yfh1 binds iron and takes it to sites of heme and Fe-S
cluster synthesis. Excess iron is trafficked to the
vacuole where it is transported across the vacuolar
membrane by Ccc1 for storage. Release of iron from
the vacuole is accomplished by Fet5/Fth1 which are
homologous to Fet3/Ftr1 (Fig 1).9 Studies of yeast
proteins such as Fet3, Yfh1, Ccc2, Atx1, and other
proteins have brought considerable insight to the
functions of their human homologs. There are
additional levels of complexity in human iron
metabolism that are caused by the additional
transport, absorption, and regulation steps that are
required in a multicellular organism. As a result, a
regulatory hormone such as hepcidin, a storage
protein like ferritin, and a transport protein like Tf do
not have homologs in yeast. As shown in Figure 2,
however, some aspects of iron uptake and utilization
are well conserved between yeast and human cells.
DIETARY IRON UPTAKE IN HUMANS
Bioavailability of Iron
In food, iron is found in 2 basic forms: inorganic
iron (both Fe(II) and Fe(III)) and heme where iron
is complexed to protoporphyrin IX. In an average
diet, inorganic iron accounts for approximately
90% of total dietary iron content, whereas heme
makes up the remaining 10%.10,11 Iron absorption
varies significantly with diet composition, iron
status of the individual, and, most importantly,
bioavailability of the different iron forms. It has
been estimated that in the developed world, where
meat consumption is relatively high, more than half
106 BLEACKLEY ET AL

Fig 1. Iron metabolism in the yeast S cerevisiae. Starting at the bottom and going clockwise, the organelles within the yeast cell are the
nucleus, the Golgi apparatus, the vacuole, and the mitochondrion. Yeast proteins are indicated by beads containing the abbreviated protein
name. Involvement of a putative intracellular chaperone is indicated with the 2 question marks. Specific transporter proteins are shown as
beads containing a channel. The oxidation state of iron and copper is indicated as suffixes. Three common uptake pathways are shown
involving Fet3/Ftr1, Smf1, and Fet4. Further details of the yeast proteins can be found in the text and at http://www.yeastgenome.org.

of all absorbed iron comes from the heme in
hemoglobin or myoglobin in dietary meats,
whereas the rest of the world obtains iron mostly
as Fe(III) from plant sources.10-14 An alternative
iron source in some diets comes from pharmaceu-
tical iron supplements such as Fe(II).12,13 Among
all natural iron sources, heme is the most bioavail-
able and its level of absorption remains unaffected
regardless of diet composition. In contrast, bioa-
vailability of inorganic iron is dependent on other
dietary components. Enhancers, such as ascorbic
acid, increase inorganic iron bioavailability by
promoting the reduction of Fe(III) to soluble Fe
(II). Inhibitors such as phytates in cereal and
polyphenols in plants form insoluble complexes
with inorganic iron and thereby reduce its absorp-
tion.11,15 In addition, inorganic iron absorption is
influenced by the level of gastric acid secretion in
IRON IMBALANCE AND DISEASE 107

Fig 2. Iron absorption in a human duodenal enterocyte. A stylized enterocyte cell is shown with the villi-containing apical membrane at
the top and the basolateral membrane and the blood vessels at the bottom. Within the cell, the organelles surrounding the nucleus include 2
mitochondria, a recycling endosome and the Golgi apparatus. Proteins are indicated by beads containing the abbreviated protein name (see
Table 1). Transporter proteins are shown as beads containing a channel. The involvement of a putative intracellular chaperone is indicated
by the question mark. Uptake of Fe(III) involves DCytB and the transporter DMT1 together with a proton gradient (Δ [H+]). Uptake of heme
iron involves HCP1. Within the cell, iron stores are shown as ferritin (Fr). The putative functions of FPN1 and Hp are indicated by their
supplying Fe(III) to apotransferrin (Tf). See text for details.

the stomach with acidity increasing the solubility of Dietary iron absorption occurs in the proximal
inorganic iron.16 An overview of dietary iron small intestine by specialized epithelial cells called
uptake in the human gut is shown in Figure 2. duodenal enterocytes. Mechanisms for inorganic
108
iron uptake allow both Fe(II) and Fe(III) to be
imported into the enterocyte. To date, 3 pathways
for iron uptake have been identified, 2 of which are
shown in Figure 2. The third pathway involves
uptake of Fe(III) through the β3 integrin mobilferrin
pathway13,17,18; this pathway is not well character-
ized and thus has been omitted from Figure 2 but a
brief overview is presented later in this review.
Divalent Metal Transporter 1
Inorganic iron is transported across the apical
membrane of enterocytes as Fe(II) by a nonspecific
divalent metal transporter 1 (DMT1; Fig 2).
Divalent metal transporter 1 is also known as
SLC11A2, natural resistance–associated macro-
phage protein 2,19 and divalent cation transporter
1,20 and is necessary for dietary iron absorption.21
Significant reduction in DMT1 results in severe
anemia that is untreatable by oral or intravenous
iron supplement demonstrating the importance of
DMT1.19,22 Protons are required by DMT1 to drive
the transport of divalent metal ions including Fe(II),
Mn(II), and Co(II).20 The stoichiometry between
protons and divalent metal ion transported by
DMT1 is dependent on extracellular pH.23 Divalent
metal transporter 1 is predicted to have 12
transmembrane (TM) domains.20 Mutation studies
were performed to identify regions important for
DMT1 function. Amino acids near the end of TM1
and in the extracellular loop between TM1 and
TM2 were shown to interact with both divalent
cations and protons.24 A spontaneous mutation in
the DMT1 gene (G185R) is located on the polar
side of the amphipathic TM4 and is likely to have
an effect on contacts between TM domains.25 In
addition, TM4 26 and His272 in TM6 27 are
important for coupling divalent cation and proton
transport. The proton-induced inward current
obtained from Fe(II) transport studies by a H272A
DMT1 mutant suggests that proton coupling
increases DMT1 affinity for Fe(II) in addition to
providing thermodynamic force for transport.27
Multiple isoforms of DMT1 result from alter-
native splicing in exon 16 and the use of different
transcription start sites (1A or 1B) in exon 1.28,29 In
rodents, differential expression and subcellular
localization of the different isoforms are regulated
both transcriptionally and posttranscriptionally.30
There are 2 common isoforms: isoform I, which has
an additional iron response element (IRE) in the 3′
untranslated region (UTR), and isoform II.28 Iso-
BLEACKLEY ET AL
form I is proposed to be the primary functional
isoform 11 and is found mainly in the apical
membrane of epithelial cells.31 The isoform I
mRNA is stabilized by the binding of iron response
protein (IRP) to the IRE resulting in increased
expression.29,32 Motifs in the carboxy termini of
isoforms I and II have recently been found to be
responsible for protein targeting and trafficking.
Isoform I is more stable in the plasma membrane
compared to isoform II. Isoform II contains a
sequence that signals recycling of the protein back
to the plasma membrane after internalization,
whereas isoform I contains a signal that targets it
to lysosomes.33
Duodenal Cytochrome b
Duodenal cytochrome b (Dcytb) is a highly
regulated ferrireductase in the apical membrane of
duodenal enterocytes that reduces dietary Fe(III) to
Fe(II).34 The mRNA and protein expression levels
of Dcytb both increase in response to iron
deficiency and hypoxia. 34,35 Unlike the iron-
regulated transporter DMT1, Dcytb transcripts do
not contain IREs, and so the mechanism whereby
iron regulates Dcytb expression remains unknown.
It has been proposed that a transcription factor may
be involved, similar to Aft1p regulation of
ferrireductases FRE1p and FRE2p expression in
yeast.36 Over a 218–amino acid region, Dcytb
shows 40% to 50% sequence identity to sheep
cytochrome b561 (cytb561), whereas the amino-
terminal region is homologous to hemoprotein p30.
However, no homology is found between Dcytb
and ferrireductases isolated from plants and
yeast.34,36 Dcytb is predicted to contain 6 trans-
membrane domains and the sequence comparison
with cytb561 shows 4 conserved histidine residues
which are thought to be involved in heme
binding.34,37 Partial conservation of the cytb561
ascorbic acid and dehydroascorbic acid binding
sites in Dcytb suggests that Dcytb may react with
one or both of these compounds. 34 Cytb561
reduces dehydroascorbate to ascorbate by trans-
porting an electron donated by cytoplasmic ascor-
bate across the membrane; thus, Dcytb may be able
to reduce Fe(III) to Fe(II) using ascorbate as an
electron donor.38 The requirement of Dcytb for
dietary iron absorption is questionable; Dcytb null
mice do not present any iron-related disorders when
fed a normal diet21 and no mutations in human
Dcytb have been reported to date.39
IRON IMBALANCE AND DISEASE
Heme Absorption
Until recently, knowledge about heme absorption
by enterocytes was limited to the idea that heme
binds to an apical membrane protein and is absorbed
by enterocytes as an intact molecule.40-42 A specific
pathway for heme absorption is evident when
studying the import of zinc mesoporphyrin, a
fluorescent heme analog, by human intestinal
Caco2 cells.43 Our understanding of heme absorp-
tion has increased considerably with the discovery of
heme carrier protein 1 (HCP1) by cDNA suppressive
subtraction using hypotransferrinemic mice.44 The
human HCP1 gene (2097 base pair) on chromosome
17q11.1 encodes a 459–amino acid polypeptide that
is predicted to have 9 transmembrane domains and
belongs to the major facilitator superfamily of
transporter proteins (reviewed in reference 45).
Within this superfamily, HCP1 most resembles the
bacterial metal-tetracycline transporter.44 Heme
carrier protein 1 contains a highly conserved motif
(GXXSDRXGRR) between TM2 and TM3, which
is also found in the metal-tetracycline transporter and
the heme exporter protein, feline leukemia virus
receptor C. However, no known heme binding
motifs, such as CXXCH in cytochrome c, are
found in HCP1 (see references 46 and 47).
Expression of HCP1 in Xenopus oocytes and
HeLa cells results in a 2- to 3-fold increase in heme
uptake, which is abolished when the gene is silenced
by HCP1-specific siRNA or in the presence of
HCP1 antisera. The temperature-dependent and
saturable activity of HCP1 suggests an energy
requirement for heme transport.44 However, this
energy requirement is presently undefined but may
involve a proton or anion gradient similar to the
mechanism used by the major facilitator super-
family transporters (see reference 45). Heme carrier
protein 1 is regulated both transcriptionally and
posttranslationally. Expression is induced by
hypoxia, whereas subcellular localization of HCP1
appears to correlate to the iron status in the body.
Under normal conditions, HCP1 is found in both the
apical membrane and cytosol. In response to iron
deficiency, HCP1 migrates to the apical membrane,
whereas cytosolic distribution peaks with the
hepatic inorganic iron store.44 Induction of heme
oxygenase 1 expression by CdCl2 also causes an
increase in HCP1 expression and heme uptake.
Thus, it is proposed that HCP1 expression and
function are proportional to the rate of heme
109
degradation by heme oxygenase 1.48 Heme carrier
protein 1 was recently shown to play a major role in
folate transport in the presence of a proton gradient
and was given the additional name proton-coupled
folate transporter (PCFT)/HCP1.49
Alternative Iron Pathways
The third pathway for the transport of iron into
enterocytes is the integrin-mobilferrin pathway
(IMP).50 Mobilferrin is a homologue of calreticulin,
a common chaperone protein within the endoplas-
mic reticulum, whereas β3 integrin is a known
adhesion protein.13 Despite having been studied for
more than a decade, the IMP pathway remains
poorly understood. It is now known that the IMP
pathway only transports iron in an inorganic Fe(III)
state.17 It is believed that during iron deficiency,
mobilferrin is secreted into the lumen of the small
intestine along with the mucosal surface protein
mucin and the 2 proteins chelate Fe(III).13 The
soluble Fe(III)-mucin-mobilferrin complex is trans-
ported to the plasma membrane and Fe(III) is taken
up into the enterocyte by β3 integrin.51 Inside the
cytosol, the assembly of a complex of β3 integrin,
mobilferrin, flavin mono-oxygenase, and DMT1
has been proposed.13 This complex, known as
paraferritin, was shown to have ferrireductase
activity capable of reducing the newly transported
Fe(III).52 Soluble Fe(II) is then free to join the pool
of Fe(II) transported across the plasma membrane
via the DMT1 pathway.
Ferroportin
Duodenal basolateral iron export into blood is
mediated by the transmembrane protein ferroportin
1 (FPN1) that is also known as iron regulatory
transporter 1 and metal transporter protein 1.
Ferroportin was simultaneously discovered by
3 groups: one group mapped the gene through
positional cloning in zebrafish exhibiting severe
microcytic anemia,53 another performed mRNA
hybridization using hypotransferrinemic mice,54
and the third group enriched a library with IRP-1
binding.55 Ferroportin 1 is a 571–amino acid protein
with a 90% amino acid sequence identity between
mice, rats, and humans.54 Encoded by the SLC40 A1
gene,56 FPN1 is also found on the basal membrane
of the placental syncytiotrophoblast as well as in
macrophages including the Kupffer cells in the liver,
spleen, and hepatocytes.53 Ferroportin 1 is the sole
iron exporter in tissues and cells where major iron
110
flow is regulated57 including dietary iron transport
into the blood, transport of iron from mother to fetus,
and iron export after the degradation of hemoglobin
from recycled RBCs.58 Experiments in Xenopus
oocytes demonstrated the iron export ability of
FPN154; however, there is still some debate whether
an associated ferroxidase is necessary for transport.
Studies indicating a colocalization of FPN1 with a
glycosylphosphatidylinositol (GPI)-linked form of
the ferroxidase ceruloplasmin (Cp) facilitates iron
efflux from brain astrocytes59 together with the
colocalization of the ferroxidase Hp with FPN1 on
the basolateral membrane of duodenal enterocytes60
suggest that a protein complex is formed in vivo.
However, FPN1 retains iron export ability even in
the absence of a ferroxidase.53 Upregulation of
FPN1 expression may be mediated by the presence
of an IRE in the 5′UTR of the FPN1 mRNA.61 The
prepropeptide hepcidin controls blood iron levels by
binding FPN1 and inducing endocytosis and
degradation as a homeostatic mechanism.62 Cou-
pling of Tf receptor (TfR)–mediated endocytosis of
plasma iron through the intestinal basolateral
membrane with hepcidin-mediated FPN1 endocy-
tosis and degradation under iron replete or overload
conditions leads to the iron loading, and subsequent
shedding of intestinal epithelial cells into the
digestive tract at the end of their 1- to 2-day lifespan
has been hypothesized.63 Thus, iron excretion in the
stool also plays a role in iron homeostasis. Studies in
zebrafish have shown that loss of FPN1 expression
is embryonic lethal, with rescue only by supple-
mentary iron injections. However, rescued zebrafish
develop severe iron deficiency anemia as a result of
the inability to absorb dietary iron.53 Mice with total
postnatal ablation of FPN1 and intestine-specific
ablation of FPN1 both present with severe systemic
iron deficiency and iron-loaded enterocytes, provid-
ing insight into the importance of FPN1 in intestinal
iron absorption.61 The role of FPN1 in dietary iron
metabolism has shed light on previously unex-
plained cases of hemochromatosis. Ferroportin-1
mutation causing iron overload disease is now called
type IV hemochromatosis or ferroportin disease.58
Hephaestin
Hephaestin is a membrane bound multicopper
oxidase that is highly expressed in the basolateral
membrane of the duodenal enterocytes and, to a
lesser extent, in the colon, brain, spleen, lung, and
placenta.64 Originally implicated in murine sex-
BLEACKLEY ET AL
linked anemia, it was found that sex-linked anemia
mice had a 582–base pair deletion in the heph gene
resulting in a 194–amino acid truncation of the Hp
protein.64,65 Although apical uptake of iron was
normal, the deletion resulted in impaired basolateral
intestinal iron transport, causing moderate to severe
microcytic hypochromic anemia.64,66 Hephaestin
was proposed as the limiting factor in intestinal iron
export. Significant sequence homology with Cp and
the yeast homologue Fet3 (see Fig 1) led to
predictions of multicopper oxidase activity.60,67
Multicopper oxidase activity was confirmed through
complementation of the low-iron growth defect
Δfet3 yeast strains. Hephaestin is primarily expressed
in the small intestine in contrast to expression of Cp
in the liver. Similar to Fet3, Hp also possesses a C-
terminal TM domain which anchors the protein to the
basolateral membrane.68 It is known that high affinity
iron uptake in yeast is mediated by a 2-component
interaction between ferroxidase Fet3p and the iron
permease Ftr1p, providing a paradigm for mamma-
lian iron transport.69
Ferroportin-Hephaestin Interactions
A study on glioma cells and astrocytes has shown
that an additional function of multicopper oxidases
is to provide stability for FPN1 at the cell surface.70
Loss of GPI-linked Cp resulted in loss of FPN1-
mediated iron transport as well as increased FPN1
internalization and ubiquitination leading to degra-
dation. In addition, lowering the extracellular
ferrous iron levels by ferroxidases played a large
role in the stability of FPN1 at the cell surface.70
This suggests that the steep Fe(II) concentration
gradient formed by Hp may not only promote
efficient iron transport, but may also stabilize the
FPN1 transport complex at the basolateral surface.
Hephaestin colocalizes with FPN1 on the basolat-
eral surface of intestinal absorptive cells, suggest-
ing that Hp and FPN1 may interact in a similar
manner.60 It is still not known whether FPN1 is iron
specific or whether Hp association provides
specificity.71 The requirement for copper in Cp
and Hp function emphasizes the importance of
copper in dietary iron uptake and iron mobilization
from tissues.72 Currently, the role of Hp in dietary
iron uptake is not clear.73 Functional studies on the
role of Cp on iron mobilization from iron storage
cells (eg, reticuloendothelial system) showed that
ferroxidase activity generates a steep concentration
gradient of ferrous iron. This in turn allows for
IRON IMBALANCE AND DISEASE
highly efficient iron efflux, providing some insight
to the possible role of Hp in intestinal iron transport.
Hephaestin ferroxidase activity may also be
required to efficiently load exported iron onto Tf
which carries only Fe(III).
IRON IN BLOOD
Red Blood Cells
Red blood cells are the predominant form of
blood cells in humans. Mature mammalian RBCs
are fully differentiated anucleate cells that do not
possess the subcellular organelles required for
protein or lipid biosynthesis. 30 Before RBCs
reach maturity, they must gather enough iron for
hemoglobin production. Hemoglobin, the major
iron carrying metalloprotein in humans, makes up
more than 95% of the dry weight of RBCs.74 More
than two thirds of total iron in a normal human
being is bound to hemoglobin in resting condi-
tions.46 The major physiologic role of hemoglobin
is oxygen transport as hemoglobin represents the
only method of oxygen transport and delivery from
lungs to peripheral tissues and cells. In addition to
the 4 globin polypeptides, each hemoglobin
molecule contains 4 iron-containing heme prosthe-
tic groups, each of which reversibly binds an
oxygen molecule. More than 80% of the iron
transported daily by Tf is used in heme synthesis.
The majority of this iron comes from senescent
RBCs that have been erythrophagocytosed by
macrophages. 74 Iron deficiency compromises
RBC production and can result in a microcytic
hypochromic anemia. The principal regulator in the
rate of eythropoiesis is the glycoprotein hormone
erythropoietin, the broad scope of which is beyond
this review (reviewed in references 46, 75, and 76).
Transferrin
Although most of the human body's iron is found
within circulating RBCs, Tf is the primary iron
transport protein in blood. Transferrin is composed
of a single polypeptide composed of 2 homologous
globular lobes (the N-lobe and C-lobes) that are
each divided into 2 subdomains. Each lobe contains
a metal-binding cleft that is capable of binding one
iron atom. Transferrin binds Fe(III) very tightly
with a Kd of ∼10−24 mol/L at physiologic pH.77
Owing to the insoluble nature of free Fe(III) under
physiologic conditions and the potential for free
iron to catalyze superoxide radical formation via the
111
Fenton reaction, the sequestering of iron is funda-
mental to the prevention of cell toxicity.6 Once
bound to Tf, Fe(III) is also unavailable for the
growth of most pathogens.78 Human serum Tf
circulates through blood at very high concentrations
(35-50 μmol/L). However, about 70% of circulating
Tf is in an iron-deplete state and available to act as a
buffer against excess free iron.79 Transferin delivers
iron to cells via an interaction with the TfR. The
importance of these proteins in iron homeostasis
was demonstrated by using knockout mice80,81,
with gene deletions in Tf and TfR causing death
shortly after birth and in utero, respectively.
Ceruloplasmin
Ceruloplasmin belongs to the multicopper oxi-
dases family and plays a vital role in mammalian
iron homeostasis as the major multicopper ferrox-
idase in blood.82 Ceruloplasmin requires 6 copper
atoms for functionality, accounting for more than
95% of the copper in human plasma. Ceruloplasmin
is postulated to have several physiologic functions
besides ferroxidase activity, including activity as a
glutathione peroxidase, an ascorbate oxidase, and
an antioxidant, as well as roles as a copper
transporter and regulator of cellular iron concentra-
tions.83 Ceruloplasmin functions in iron home-
ostasis by controlling the rate of cellular iron
efflux.84 In the absence of Cp, cellular iron egress is
inhibited with subsequent sequestration of iron
within cells.84 This regulatory function of Cp is
connected with its role in loading apo-Tf with Fe
(III), a process that requires the catalytic oxidation
of Fe(II) released from cells.77 However, it is
perhaps its biological function within the central
nervous system that Cp is most noted. Deficiencies
in Cp result in iron accumulation within the basal
ganglia that can lead to progressive neurodegen-
erative disease.85 Aceruloplasminemia is an auto-
somal recessive disorder caused by a loss-of-
function mutation in the gene for Cp.86 Affected
individuals have a complete absence of functional
serum Cp and elevated levels of serum ferritin and
can also present with anemia, diabetes, retinal
degeneration, and neuronal defects.87 The role of
Cp in brain iron metabolism and neuronal survival
has been reviewed recently.82
Iron Recycling by Macrophages
Macrophages are phagocytic white blood cells
with roles in innate and cell-specific immunity.
112
Macrophages also play an important role in iron
recycling. Plasma iron levels are largely regulated
by the rate of iron release from macrophages.63
Macrophages extract iron from hemoglobin that has
been acquired from phagocytosed senescent RBCs.
Iron is transported into macrophages through
phagocytic vacuoles that contain a divalent metal
transporter, Nramp1, a homolog of the duodenal
transporter DMT1.88 Nramp1 transports iron to the
cytoplasm of the macrophage where it is seques-
tered until needed. Ferroportin-1, the duodenal iron
exporter, also functions to mobilize iron from
macrophages. 63 Macrophage iron recycling is
regulated by hepcidin levels. Hepcidin interacts
with FPN1 causing its internalization and degrada-
tion.89,90 The degradation of FPN1 results in iron
sequestering in macrophages.91 The iron-binding
properties of macrophages also play an important
role during infection, as iron sequestration by host
macrophages can suppress pathogen proliferation.92
IRON UPTAKE BY OTHER CELLS
Transferrin Receptor 1
Cellular iron uptake is a well-characterized, pH-
dependent process of receptor-mediated endocyto-
sis.93 The TfR is a ubiquitous protein, anchored to
cell membranes by a short cytoplasmic and TM N-
terminal region. The TfR forms a homodimer,
joined by 2 intermolecular disulfide linkages in this
anchored region. The soluble extracellular domain
of the receptor extends into the plasma and is
available for Tf binding. Fe(III)-bound Tf enters
cells by preferentially binding to the TfR with high
affinity (KD, ∼0.1-10 nmol/L).94-96 The Tf-TfR
complex is internalized by endocytosis into a
clathrin-coated pit. The endosome is acidified to
pH ∼5.6 through the action of an ATPase pump.94
At this lower pH, Fe(III) is released from Tf,
whereas apo-Tf remains bound to the receptor. The
removal of Fe(III) from Tf is a complex and
incompletely understood process involving salt
concentration, pH, and a potential unknown
chelator inside the endosome.97 Upon release, it is
thought that Fe(III) is reduced to Fe(II) by the
ferrireductase Steap3.98 The Fe(II) can then be
transported across the endosomal membrane by
DMT1 and released into the intracellular environ-
ment.99 The Tf-TfR complex is recycled to the
outside of the cell and apo-Tf is released. The entire
process of cellular iron uptake from extracellular
BLEACKLEY ET AL
receptor binding to endosomal release occurs in less
than 3 minutes.93
Lactoferrin and Melanotransferrin
The role of Tf homologs in iron delivery has
always been poorly understood. Lactoferrin (Lf)
and melanotransferrin (MTf) share 37% to 39%
sequence similarity with Tf. Unlike soluble Tf, the
majority of MTf is tethered to the cell membrane by
a GPI anchor.100 However, a small amount of
soluble MTf (sMTf) exists in serum, saliva,
cerebrospinal fluid, and urine.101 Another differ-
ence between Tf and MTf occurs with iron binding;
MTf contains only one Fe-binding site.102 The
tissue expression patterns of MTf and sMTf are
quite different from Tf and TfR.101 Melanotrans-
ferrin was originally identified at high levels in
melanoma cells and fetal tissue.101,103,104 More
recently, MTf has been found highly expressed in
an array of tissues including brain, salivary gland
epithelium, pancreas, testis, kidney, and sweat
gland ducts.105 The most significant difference
between these homologs and Tf is that neither
membrane-bound MTf101 nor sMTf106 has been
shown to bind to TfR1 and deliver iron to cells.
Knockout mice, with a gene deletion for MTf,
showed no morphological, histologic, behavioral,
hematological, or Fe status variations compared to
wild-type mice.107 Although several hypotheses
have been proposed for potential MTf functions,107
it is probable that MTf does not play a significant
role in Fe transport and delivery.108
Lactoferrin, a soluble homologue of Tf, pos-
sesses both Fe-binding sites and is able to bind Fe
(III) with a higher affinity than Tf.109 Human Lf is
expressed in glandular epithelial cells and found in
breast milk and other mucosal secretions.110 There
exists some debate regarding the role of the Lf in
iron metabolism. Like the MTf knockout, deletion
of the Lf gene results in no change in phenotype.111
Although Lf has been postulated to have a function
in neonate iron absorption, it is generally believed
to have a larger role in immunity acting as an
antimicrobial agent.110
INTRACELLULAR IRON METABOLISM
Once taken into a cell, iron has 2 possible fates:
incorporation into iron proteins usually as heme or
Fe-S clusters, or storage in ferritin for later use
during iron deficiency. Intracellular iron proteins
have a wide range of functions. Cytochromes
IRON IMBALANCE AND DISEASE
including those in the electron transport chain and
cytochrome P450s in the oxidative metabolism of
drugs and other compounds112 are heme proteins.
Aconitase, the enzyme responsible for isomeriza-
tion of citrate to aconitate, is an Fe-S protein,
whereas other Fe-S proteins including hydroge-
nases act as electron carriers. Ribonucleotide
reductases, the enzymes that control the pool of
deoxyribonucelotides for DNA synthesis, are
examples of nonheme iron proteins.113 Nonheme
iron proteins contain iron but not complexed in
heme or Fe-S clusters.
Mitochondrial Iron Metabolism
Both heme and Fe-S clusters are synthesized in
mitochondria making this organelle a major target for
intracellular iron metabolism. Mitoferrin, encoded
by the mfrn gene, is a mitochondrial membrane
protein responsible for iron uptake into the mito-
chondria for heme biosynthesis during eythropoi-
esis.114 A second gene, mfrn2, encodes a similar
protein that is sufficient to provide basal level of iron
required by nonerythroid cells.115 Characterization
of the orthologous pair MRS3/4 in S cerevisiae
provided much of the basis for the identification and
characterization of mfrn/mfrn2.116-119 Inside the
mitochondria, iron is bound by the mitochondrial
iron chaperone frataxin (FXN),120 a protein that
forms multimers with iron stored in the core.121
Frataxin is involved in mitochondrial iron storage
while preventing the formation of damaging free
radicals,122-124 maintaining a pool of Fe(II) in the
mitochondria, and delivering iron to sites of heme
and Fe-S cluster assembly.125-127 Monomeric FXN
has been shown to bind iron and interact with
proteins involved in heme and Fe-S cluster synthesis
providing an alternate model to the multimeric model
previously described.128 Mutations in the FXN gene
cause the neurodegenerative disorder Friedreich's
ataxia (FRDA). 129 Studies of the yeast FXN
homolog Yfh1 have contributed significantly to the
understanding of the role of FXN in mitochondrial
iron metabolism. Heme and Fe-S cluster synthesis
are both complicated procedures, and have been
reviewed recently.130-133
Ferritin
Ferritin is the major intracellular iron storage
protein in animals and prokaryotes. Vertebrate
ferritin, which is expressed ubiquitously, is a
hollow sphere composed of 24 subunits of H and
113
L types in varying ratios depending on the type of
cell and physiologic conditions. Iron is stored in the
lumen of the multisubunit sphere. Up to 4500 iron
atoms can be stored in each ferritin multimer. As
iron is transported into the ferritin multimer core, it
is oxidized from Fe(II) to Fe(III) by the H subunit
allowing formation of nonreactive ferrihydrate
which assembles as a solid in the core in a process
involving the L-subunit.134-136 Although ferritin is
a predominantly cytoplasmic protein, recent evi-
dence has shown that it can also be found in the
nucleus137 and mitochondria.138 A small quantity
of glycosylated ferritin is secreted into the
serum.139 Cytoplasmic ferritin has been shown to
associate with microtubules.140 Studies into the
distribution of microtubule-associated ferritin com-
bined with evidence that association with micro-
tubules increases ferritin iron binding in vitro
support a role for microtubules in the distribution
of intracellular ferritin and thus iron metabolism as
a whole.141
Regulation of Iron Metabolism
The cell must regulate all stages of iron
metabolism including uptake, storage, and utiliza-
tion. In vertebrates, expression of proteins such as
TfR and ferritin is regulated by cellular iron levels at
the posttranscriptional level.142 Briefly, mRNAs of
some proteins of iron metabolism contain IREs in
the UTRs. Iron response elements are 30-nucleotide
hairpin structures that are conserved in vertebrates
and some insects and bacteria. Under iron-starved
conditions 2 IRP1 and IRP2 bind to the IREs. In
mRNAs for proteins involved in iron uptake such as
TfR, the IREs are found in the 3′UTR; IRP binding
to IREs in the 3′UTR stabilizes the mRNA
increasing translation of the proteins. Proteins
such as ferritin that are involved in sequestering
iron have IREs in the 5′UTR; IRP binding to 5′IREs
results in translational inhibition and decreased
synthesis of the proteins. RNA binding activity of
IRP1 is regulated by the formation of an Fe-S cluster
which inhibits IRE binding in iron replete condi-
tions.143 The IRE/IRP regulation of iron metabolism
has been reviewed recently.144,145
Intracellular Iron Transport
Intracellular iron transport between sites of
uptake, storage, and utilization is not well under-
stood. Iron that is not complexed into proteins, not
stored in ferritin, nor localized to the mitochondria
114
has been termed the labile iron pool (LIP). The
oxidation states of iron in the LIP remain unclear.
Evidence supports the hypothesis that the LIP is
bound to low-molecular-weight proteins as
opposed to small molecules such as citrate.146,147
Intracellular copper transport occurs via a number
of copper metallo-chaperone proteins.148-150 From
these 2 lines of evidence, some have hypothesized
that intracellular iron is transported via protein
chaperones in a manner similar to copper, and it is
these small iron chaperone proteins that are being
detected in the analysis of the LIP. In erythroid
cells, where 85% of heme synthesis occurs,
inhibition of endosome mobility decreases the
incorporation of 59Fe into heme, supporting the
hypothesis that there is a direct interaction between
iron-loaded vesicle and the mitochondria in
RBCs.130,151 However, vesicular trafficking cannot
be the only form of intracellular iron transport. In a
S cerevisiae strain with a deletion of end4 which is
defective in endocytosis, iron is transported from
the cytosol to the vacuole for storage (S cerevisiae
lack an expressed ferritin gene and store iron in the
vacuole).152 Thus, another intracellular iron trans-
port mechanism must exist, and this remains the
subject of intense investigation.
REGULATION OF IRON ABSORPTION
Hepcidin and Hemojuvelin
Hepcidin is synthesized by the liver as a
prepropeptide consisting of 84 amino acid residues.
Removal of a 24–amino acid signal peptide and
cleavage at Arg-59 by furin result in the release of
the active peptide hormone that is 25 amino acids in
length.153 Hepcidin is rich in cysteine residues and
forms a hairpin with β-sheet structure stabilized by 4
disulfide bonds.154 Hepcidin was identified as a
negative regulator of iron absorption based on iron
level and distribution in hepcidin-deficient or
-overexpressing mouse models.155,156 Hepcidin
downregulates iron absorption by binding FPN1
inducing its internalization and degradation.62 After
hepcidin incubation, FPN1 is phosphorylated at 2
adjacent tyrosine residues (T302-T303) in an
extracellular loop between TM6 and TM7.157 The
trafficking of internalized FPN1 through multi-
vesicular body to lysosomes for degradation
involves the protein complex known as the Endo-
some Sorting Complex Required for Transport. As a
secondary effect of FPN1 downregulation, increased
BLEACKLEY ET AL
intracellular iron level lowers the expression of
DMT1, resulting in a decrease in iron uptake.35,158
Hepcidin expression is modulated at the tran-
scription level by multiple factors including inflam-
mation, Tf saturation, iron deficiency, and hypoxia.
In response to inflammation, the cytokine inter-
leukin-6 interacts with its receptor on hepatocyte
membranes and activates Stat3, which upregulates
hepcidin expression by binding to a regulatory
element on the hepcidin gene.159-161 A better
characterized pathway for regulation of hepcidin
expression involves the interaction of hemojuvelin
(HJV) with the bone morphogenetic protein (BMP)/
Smad signaling pathway. 162 Hemojuvelin is
expressed by liver, heart, and skeletal muscles,163
and is found in 2 forms: GPI-anchored HJV (mHJV)
and soluble HJV (sHJV).164 The 2 forms recipro-
cally regulate hepcidin expression in response to the
iron requirement of the body. As a BMP coreceptor,
mHJV interacts with BMP2/BMP4, enhancing
BMP binding to type I and type II receptor serine/
threonine kinases. Type II receptor kinase activates
type I receptor kinase, which in turn phosphorylates
receptor-regulated Smad. Phosphorylated receptor-
regulated Smad complexes with Smad4 and the
complex translocates into the nucleus promoting
hepcidin transcription.165,166 Soluble HJV com-
petes for binding to BMP2/BMP4 against mHJV to
inhibit BMP/Smad signaling and, hence, blocks
hepcidin production.159,166
Hepcidin expression is determined by the relative
levels of mHJV and sHJV. Expression of both forms
of HJV depends on the level of Tf-bound iron but not
other chelated iron.166 The release of mHJV from
membrane upon interaction with its receptor,
neogenin, is suggested to downregulate hepcidin
expression and is inhibited by holo-Tf.167 Contrarily,
iron deficiency and hypoxia stimulate sHJV produc-
tion by stabilizing hypoxia inducible factor-1 α.
Hypoxia inducible factor-1 α induces the expression
of furin, which cleaves HJV at Arg-335 and releases
the mature sHJV into cell culture media.168
Hemochromatosis Protein
The precise role of hemochromatosis protein
(HFE) in the regulation of iron absorption remains
to be elucidated. Hemochromatosis protein is
expressed ubiquitously at low levels in tissues169
and is highly expressed in intestinal crypt cells and
liver.170 The level of HFE expression inversely
correlates to the level of iron uptake.171 The
IRON IMBALANCE AND DISEASE
absence of HFE is associated with iron overloading,
whereas overexpression of HFE reduces iron
absorption (reviewed in reference 172). The HFE
polypeptide is composed of 3 extracellular loops
(α1, α2, α3), a TM domain, and a short cytosolic
tail. Its 3-dimensional structure is stabilized by 2
disulfide bonds, one of which gives rise to the α3
loop for noncovalent interaction with β2 micro-
globulin (β2M).173,174 The HFE-β2M interaction is
essential for HFE translocation across the plasma
membrane for cell surface expression of the
complex. 174 The HFE-β2M complex interacts
with the helical domain of TfR1.95,175,176 Coloca-
lization95 and interaction177 with TfR2, which is
selectively expressed in crypt cells, have also been
reported recently.
Hemochromatosis protein is believed to act as a
sensor of the iron status in the body. Various models
regarding HFE-mediated regulation of iron home-
ostasis have been postulated. Studies have indicated
that HFE sequesters TfR1 when Tf saturation falls
within the normal range39 but competes with holo-Tf
for binding to TfR1 with increased Tf saturation.178
Thus, HFE is proposed to sense the body's iron
requirement based on the level of Tf saturation with
iron. The original model assumed that HFE forms a
heterotrimer with β2M and TfR1 in the basolateral
membrane of enterocytes and signals expression of
iron transport proteins through the action of IRP,179
or in the plasma membrane of hepatocytes to
modulate iron absorption by inducing hepcidin
production.180 A recent study demonstrated the
interaction between HFE and TfR2.177 Because
TfR2 is distributed in lipid rafts, it is believed to play
a role in signal transduction upon holo-Tf binding. In
stimulated erthroleukemic cells, TfR2 activates
extracellular signal-regulated kinases 1 and 2 and
p38 mitogen-activated protein kinases.181 The latest
model for the mechanism of action of HFE, again,
presumes HFE-β2M to be involved in sensing Tf
saturation and is complexed to TfR1 under normal or
low serum iron condition. As Tf saturation increases,
competitive binding to TfR1 occurs between HFE
and holo-Tf. The resulting free HFE-β2M then binds
TfR2 to signal hepcidin production via the extra-
cellular signal-regulated kinase/mitogen-activated
protein kinase pathway.181,182 However, the require-
ment for HFE in TfR2-mediated signal transduction
is unknown.177 Colocalization of HFE-β2M with
DMT1 in apical membrane of enterocytes during
iron deficiency implies that HFE may function to
115
regulate duodenal iron absorption directly.171 Ecto-
pic expression of HFE increases HFE-β2M apical
distribution, which in turn reduces the rate of apical
iron absorption.183
DISEASES OF IRON IMBALANCE
Divalent Metal Transporter 1 Deficiency
Mutations of transporter proteins involved in
dietary iron uptake result in anemia. DMT1
mutations are autosomal recessive and cause
hypochromic microcytic anemia and hepatic iron
accumulation. Defective DMT1 loses its function
owing point mutations, in-frame deletion of a single
amino acid, or the complete deletion of an exon due
to alternative splicing. Mutations that give rise to the
exon skipping DMT1 variants do not completely
remove DMT1 function.184-186 DMT1 exon 12
skipping normally occurs at low levels (∼10%
mRNA); however, the DMT1 G1285C mutation
increases the tendency of exon skipping to ∼90% of
DMT1 mRNA, giving a nonfunctional splice variant
that lacks TM8. The remaining full-length mRNA
encodes a fully functional mutant DMT1, where a
glutamate is replaced by aspartate in an intracellular
loop. The small amount of functional DMT1 is
thought to be responsible for hepatic iron overload
observed in the patients.187 As discussed previously,
PCFT/HCP1 is shown to be involved primarily in
folate absorption rather than heme absorption. No
mutation in PCFT/HCP1 that affects heme uptake
has been identified to date. However, mutation of
PCFT/HCP1 has been shown to lower the stability
or affect membrane trafficking of the protein,
impairing folate dietary absorption and transport to
central nervous system. Folate deficiency leads to
erythroblast apoptosis, resulting in megaloblastic
anemia from compromised erythropoiesis.74,188
Hemochromatosis
Hereditary hemochromatosis is a primary iron
overload disease with consequent tissue damage
from iron-induced oxidative stress. Hereditary
hemochromatosis is caused by one or more
mutations in genes encoding proteins that are
involved in maintaining iron homeostasis. Hemo-
chromatosis type I is caused by mutations in HFE
with most patients being homozygous for the
C282Y mutation. C282Y disrupts a disulfide
bond required for the formation of the α3 domain,
disrupting HFE-β2M interaction and thereby
116
inhibiting surface expression of the protein.169 Not
all individuals who are homozygous for HFE
C282Y manifest iron overloading, suggesting
incomplete penetrance of this mutant. 172 Iron
overloading due to mutations in HJV or hepcidin
is known as hemochromatosis type II or juvenile
hemochromatosis. Juvenile hemochromatosis is a
severe form of hereditary hemochromatosis and is
characterized by an early onset, usually before
30 years of age. The majority of type II cases are
caused by mutations in HJV. A rare form of
hemochromatosis type III is associated with muta-
tions in TfR2 which is implicated in regulation of
iron transport by interacting with HFE. Patients
with one of the 3 types of hemochromatosis
discussed above show autosomal recessive inheri-
tance and are found to have abnormally low levels
of hepcidin, suggesting that these mutations act
through a common mechanism.189 Unlike hemo-
chromatosis types I, II, and III, hemochromatosis
type IV results from mutations in FPN1 and is also
known as ferroportin disease.71,190 Ferroportin-1
mutations display autosomal dominant inheritance
and are heterozygous in clinical presentation. All
FPN1 mutations reported to date involve either a
single base change or a deletion. Recently, FPN1
has been characterized as a homodimer which
probably explains in part the autosomal dominant
nature of ferroportin disease as the mutant subunit
may affect the assembly of the dimer and possibly
the subsequent membrane localization or response
to hepcidin regulation.191
Aceruloplasminemia
Aceruloplasminemia is an iron metabolic dis-
order caused by recessive mutations in the Cp gene
that result in a complete absence of Cp ferroxidase
activity. Some mutations in Cp cause premature
termination of Cp translation. The resulting trun-
cated product is not or partially copper loaded.192
Patients with aceruloplasminemia are anemic
owing to the low serum iron concentration and
high serum ferritin level. Despite the low serum
iron level, iron accumulates in the brain and visceral
organs such as the liver and pancreas. Excess iron
deposition in the brain leads to loss of neurons in
basal ganglia and Purkinje cells in cerebellar
cortex.193 As discussed in detail earlier, Cp is a
multicopper ferroxidase that is synthesized by
astrocytes in the mammalian central nervous
system as a GPI-anchored protein. Colocalization
BLEACKLEY ET AL
with FPN1 on astrocyte surface suggests a role in
iron transport in the brain.59 Ceruloplasmin is
essential for brain iron metabolism and neuronal
survival and functions as an antioxidant by
preventing Fe(II)-induced lipid peroxidation and
reactive oxygen species (ROS) generation. The
level of iron accumulation parallels the extent of
astrocyte deformity and formation of globular
structures within these cells. Fe(II)-stimulated free
radical production and lipid peroxidation, as
indicated by a significant increase in the levels of
4-hydroxynonenal and malondialdehyde (markers
of lipid peroxidation) in astrocytes, cause glial cell
death. The loss of glia function lowers the survival
of neurons and accentuates oxidative neurodegen-
eration with Fe(II)-induced oxidative stress.192
NEURODEGENERATIVE DISEASES RESULTING
FROM THE LOSS OF REGULATION OF
INTRACELLULAR IRON
FriedreichTs Ataxia
Friedreich's ataxia is an autosomal recessive
neurodegenerative disease characterized by degen-
eration of Purkinje neurons of the cerebel-
lum.194,195 It is the most common hereditary
ataxia among whites with a prevalence of 1 to 2
in every 50 000 people.196 The majority (∼97%) of
patients with FRDA are homozygous for an
abnormal expansion of GAA triplet repeats in
intron 1 of the FXN gene, whereas the remainder
are compound heterozygotes for a point mutation or
microdeletion in one allele and a GAA repeat
expansion in the other.197 The extra repeats in the
FXN gene are shown to interfere with transcription
by forming a triplex DNA structure called “sticky
DNA,” and the size of the repeats parallels the time
of onset and the rate of FRDA progression.198
Frataxin deficiency causes iron accumulation in
the mitochondria, reduction in iron-sulfur cluster–
dependent enzymes, and altered antioxidant
defense. 195 The excess iron in mitochondria
displaces manganese in superoxide dismutase and
renders the enzyme inactive.199 Superoxide dis-
mutase inactivity causes oxidative stress and lipid
peroxidation in patients with FRDA, as indicated
by increased levels of urinary 8-hydroxy-2′-deox-
yguanosine, a marker for oxidative DNA damage,
and plasma malondialdehyde, respectively.198 The
effect of decreased frataxin is not limited to the
mitochondria as FXN deletion in mouse tissues is
IRON IMBALANCE AND DISEASE
recently shown to impair extramitochondrial iron-
sulfur proteins.200
ParkinsonTs Disease
Parkinson's disease results from the death of
dopamine-producing neurons in substantia nigra.
Parkinson's disease is characterized by the pre-
sence of insoluble fibrillar structures, called Lewy
bodies, in the presynaptic nerve terminals.201 A
major component of Lewy bodies is α-synuclein, a
small acidic protein of 140 amino acid residues.202
Native α-synuclein displays an unfolded random
coil structure. Interaction with factors such as
proteins, lipids, oxidants, or metal ions promotes
α-synuclein folding and aggregation, leading to
Lewy body formation.203 A potential IRE in the 5′
UTR of α-synuclein has been described, indicating
that iron may also function to regulate α-synuclein
production at the translational level.204 Iron
accumulates at the site of substantia nigra degen-
eration in patients with Parkinson's disease.205 The
precise mechanism through which iron promotes
neurodegeneration is not known. However, it has
been postulated that iron interacts with α-synuclein
to induce its oligomerization through the genera-
tion of free radicals, resulting in Lewy body
formation.206 A recent study has also shown that
Fe(III) promotes the production of short and thick
fibrils when it interacts with both wild-type and
mutant α-synuclein.203 Although harmful, iron
cannot be eliminated entirely from these dopami-
nergic cells because iron is required for tyrosine
hydroxylase activity during the synthesis of
dopamine; iron is also involved in the conversion
of excess dopamine to neuromelanin. However,
when bound to excess iron, neuromelanin acquires
ferrireductase activity and converts Fe(III) to Fe(II)
which is released owing to its poor affinity for
neuromelanin. Iron-induced oxidation of dopamine
releases hydrogen peroxide, which reacts with Fe
(II) via the Fenton reactions to generate harmful
radicals which can cause cell death.205
Alzheimer's Disease
The main features of Alzheimer's disease are the
formation of senile neuritic plaques and neurofi-
brillary triangles. Senile neuritic plaques are
composed of insoluble α-amyloid peptide (Aα)
which aggregates spontaneously in the presence of
divalent metals, such as Zn(II), Cu(II), and Fe
(II).207 Aα is a cleavage product of amyloid β
117
precursor protein (APP) whose translation may be
regulated by intracellular iron levels via a putative
IRE in the 5′UTR of APP.208 At low concentra-
tions, Aα exhibits antioxidant activity; however,
Aα is toxic to the cells at high concentrations.
Accumulation of Aα in patients with Alzheimer's
disease is thought to be caused by increased APP
production or inefficient clearance of Aα. Fe(III)
binds to Aα at high affinity and induces its
aggregation, whereas heme binds and prevents Aα
aggregation. Fe(III)-bound Aα plaques possess
high reduction potential and reduce Fe(III) to Fe
(II) at high efficiency. The reduced iron reacts with
trapped oxygen in the senile plaques to ROS via
Fenton reactions. Similarly, the heme-Aα complex
functions as a peroxidase to produce ROS.209,210
Met35 of Aα is believed to serve as an electron
donor in the generation of hydrogen peroxide from
oxygen as oxidized-Met35-Aα is isolated from
amyloid plaques. The removal of hydrogen per-
oxide inhibits Aα neurotoxicity, suggesting that Aα
plaques cause neurodegeneration through ROS
production. As a result of free radical reactions,
irreversible dityrosine linkages are formed between
Aα. Thus, it has been hypothesized that toxicity
may require oxidative modification of Aα.211
Neuroferritinopathy
Neuroferritinopathy is a cognitive and motor
disorder caused by neuronal degeneration and free
radical toxicity from iron and ferritin deposition in
the brain, particularly the basal ganglia. Neurofer-
ritinopathy is a dominant disorder in which patients
have a single nucleotide insertion in the carboxyl
terminal of the ferritin light chain. Insertion of an
adenine at position 460 to 461 or thymine/cytosine
at position 498 to 499 in exon 4 of the ferritin light
chain results in a frame shift causing formation of a
new stop codon that is downstream of the normal
position. Consequently, this replaces the last 22 or 9
amino acids with a different 26 or 25 amino acids,
respectively.212,213 Based on protein modeling data,
the C-terminal of ferritin light chain is predicted to
be involved in pore formation in the ferritin
dodecahedron complex. The mutant C-terminal
sequence likely affects ferritin subunit interaction
and stability, and thereby increases iron availability
and the cell's sensitivity to oxidative stress.214 The
exact mechanism through which mutations in
ferritin light chain induce neurodegeneration is
undefined. However, it has been postulated that the
118
incorporation of mutant light chains partially
compromises the structure and function of ferritin.
Iron and ferritin aggregation suggests that the
mutant subunit may promote the release of iron
by ferritin, leading to oxidative stress and cell
death.205 It has been shown that ferritin light chain
level is not critical in the pathogenesis of
neuroferritinopathy as neurodegeneration is unde-
tectable in an individual with decreased ferritin
light chain expression due to a heterozygous start
codon mutation.215
CONCLUDING REMARKS
Because of its roles in oxygen transport, the
respiratory chain, and deoxyribonucleotide bio-
synthesis, iron is an essential element in blood cells.
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