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REGULATION OF
INTRACELLULAR IRON METABOLISM
Tracey A. Rouault, MD, Head, Section on Human
Iron Metabolism Manik
Ghosh, PhD, Senior Fellow Esther
Meyron-Holtz, PhD, Senior Fellow Sharon
Cooperman, MD, Postdoctoral Fellow Kuanyu
Li, PhD, Postdoctoral Fellow Fanis
Missirlis, PhD, Postdoctoral Fellow Helge
Uhrigshardt, PhD, Postdoctoral Fellow Emine
Yikilmaz, PhD, Postdoctoral Fellow Wing
Hang Tong, PhD, Staff Scientist William
Land, BS, Technician Sara
Holmberg, BS, Postbaccalaureate Fellow Cindy Yang, BS, Postbaccalaureate Fellow |
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Previously, the laboratory identified and characterized
the major iron metabolism–regulatory system in mammals. Iron-dependent
alterations in expression of iron metabolism genes such as ferritin and the
transferrin receptor are mediated mostly by post-transcriptional mechanisms.
Iron-responsive elements (IREs) are RNA stem-loops found in the 5´ end of
ferritin mRNA and the 3´ end of transferrin receptor mRNA. We have
identified, cloned, expressed, and characterized two essential iron-sensing
proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2
(IRP2). IRPs register changes in cytosolic iron levels and bind to IREs when
iron levels are depleted, resulting in the inhibition of translation of ferritin
mRNA and other transcripts with IREs near the 5´ end as well as extension of
the half-life of the transferrin receptor mRNA and possibly other mRNAs. Much
of our work involves elucidating the mechanisms of IRP function and
determining the physiologic consequences of misregulation of iron metabolism.
Our studies have led to investigations of the role of iron metabolism
misregulation in human diseases such as Parkinson’s disease, hemochromatosis,
infant Gracile syndrome, Friedreich’s ataxia, and severe iron deficiency
anemia. Iron-sulfur
cluster assembly Tong, Li, Uhrigshardt IRP1 is an iron-sulfur protein related to
mitochondrial aconitase, a citric acid cycle enzyme that functions as a
cytosolic aconitase in cells replete with iron. Regulation of the RNA binding
activity of IRP1 involves a transition to a form that loses both iron and
aconitase activity from a form of IRP1 in which a [4Fe-4S] cluster is bound.
The [4Fe-4S]-containing protein does not bind to IREs, and the status of the
cluster appears to determine whether IRP1 will bind to RNA. Recently, we
identified mammalian enzymes of iron-sulfur cluster assembly that are
homologous to the NifS and Nif U genes implicated in bacterial
iron-sulfur cluster assembly. We showed that these gene products facilitate
assembly of the iron-sulfur cluster of IRP1. We discovered that single genes
in the human genome encode mitochondrial and cytosolic forms of the cysteine
desulfurase IscS and the proposed scaffold proteins IscU and NFU. NFU is an
abundant protein in mitochondria and cytosol that assembles a [4Fe-4S]
cluster. NFU may function as a scaffold for iron-sulfur cluster assembly by
donating its newly assembled clusters to recipient proteins. In the human
disease Friedreich’s ataxia iron-sulfur cluster biogenesis is impaired; our
studies aim to elucidate the role of frataxin in mammalian iron-sulfur
cluster assembly. Missirlis F, Hu J, Kirby K, Hilliker AJ,
Rouault TA, Phillips JP. Compartment-specific protection of iron-sulfur
proteins by superoxide dismutase. J
Biol Chem 2003;278:47365-47369. Tong WH, Jameson GN, Huynh BH, Rouault TA.
Subcellular compartmentalization of human Nfu, an iron-sulfur cluster
scaffold protein, and its ability to assemble a [4Fe-4S] cluster. Proc Natl Acad Sci USA
2003;100:9762-9767. Iron-dependent
degradation of IRP2 and other proteins Ghosh IRP2 also binds to IREs in iron-depleted cells
but, unlike IRP1, it is degraded in cells that are replete with iron.
Experimental evidence indicates that IRP2 undergoes iron-catalyzed oxidation.
The oxidized protein is then selectively ubiquitinated and degraded by the
proteasome. Indirect evidence suggests that the degradation pathway of
numerous other proteins involves oxidative modification followed by
ubiquitination and proteasomal degradation of the ubiquitinated substrate.
Heme has been implicated in IRP2 degradation, but it is not yet clear whether
free heme directly oxidizes IRP2 or whether heme is a cofactor for a trans-acting factor involved in
iron-dependent degradation. Bourdon E, Kang DK, Ghosh M, Drake SK, Wey J,
Levine RL, Rouault TA. The role of endogenous heme synthesis and degradation
domain cysteines in cellular iron-dependent degradation of IRP2. Blood Cells Mol Dis 2004;31:247-255. Kang DK, Jeong J, Drake SK, Wehr N, Rouault
TA, Levine RL. Iron-regulatory protein 2 as iron sensor: iron-dependent
oxidative modification of cysteine. J
Biol Chem 2003;278:14857-14864. Yamanaka K, Ishikawa H, Megumi Y, Tokunaga F,
Kanie M, Rouault TA, Morishima I, Minato N, Ishimori K, Iwai K.
Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2.
Nat Cell Biol 2003;5:336-340. Physiology
and regulation of iron metabolism Cooperman, Meyron-Holtz, Land, Missirlis,
Holmberg, Yang To study the physiology of iron metabolism, we
generated loss-of-function mutations of IRP1 and IRP2 in mice through
homologous recombination in embryonic cell lines. In the absence of
provocative stimuli, there are no abnormalities in iron metabolism associated
with loss of IRP1 function. IRP2-/-mice develop a progressive
movement disorder characterized by gait abnormalities and a Parkinsonian
tremor. Animals accumulate ferritin iron in axons and develop axonal degeneration.
These findings are greatly accentuated in animals that lack one copy of IRP1
in addition to both copies of IRP2. Thus, IRP2 is the predominant regulator
of post-transcriptional iron metabolism in animals, but IRP1 also contributes
to baseline regulation. Ferritin iron accumulations in the brain can be
detected on magnetic resonance imaging. Vacuolar changes that develop as a
result of neuronal cell body loss in regions such as the substantia nigra are
detectable histopathologically and correlate with decreased T2 signals on
MRI. Animals that lack both IRP1 and IRP2 do not survive past the blastocyst
stage. To deconvolute the contribution of specific iron metabolism proteins
to the neurodegeneration of IRP2-/- mice, we generated and are now
analyzing transgenic mice that overexpress ferritin subunits and transferrin
receptor. To analyze the role of iron metabolism in neurodegeneration more
simply, we developed the Drosophila
model system and discovered that overexpression of ferritin leads to adult-onset
neurodegenerative disease. The discovery that misregulation of iron
metabolism leads to neurodegeneration in mice and flies has increased our
interest in the role of iron metabolism abnormalities in human
neurodegeneration. Accordingly, we plan to study selected humans with
Parkinson’s disease and related neurodegenerative diseases by sequencing
candidate disease genes and making use of the new Meyron-Holtz EG, Ghosh MC, Iwai K, LaVaute T,
Brazzolotto X, Berger UV, Land W, Ollivierre-Wilson H, Grinberg A, Love P,
Rouault TA. Genetic ablations of iron regulatory proteins 1 and 2 reveal why
iron regulatory protein 2 dominates iron homeostasis. EMBO J 2004;23:386-395. Rouault TA. Hepatic iron overload in alcoholic
liver disease—why does it occur and what is its role in pathogenesis? Alcohol 2003;30:103-106. Rouault TA. How mammals acquire and distribute
iron needed for oxygen-based metabolism. PloS
Biol 2003;1:326-328. Smith SR, Cooperman S, Lavaute T, Tresser N,
Ghosh M, Meyron-Holtz E, Land W, Ollivierre H, Jortner B, Switzer R, Messing
A, Rouault TA. Severity of neurodegeneration correlates with compromise of
iron metabolism in mice with iron regulatory protein deficiencies. Ann NY Acad Sci 2004;1012:65-83. Wu LJ, Leenders AGM, Cooperman S, Meyron-Holtz
E, Smith SR, Land W, Tsai RYL, Berger UV, Sheng ZH, Rouault TA. Expression of
the iron transporter ferroportin in synaptic vesicles and the blood brain
barrier. Brain Res
2004;1001:108-117. Structural
characterization of IRPs and IREs Yikilmaz We have purified milligram quantities of IRP1
and IRP2 by overexpression in Pichia
pastoris and are working on crystallization of each IRP. In addition, we
are attempting to co-crystallize each IRP in a complex with IRE. We also
characterized and overexpressed an IRP-like protein from Plasmodium falciparum. To ensure that high-quality IRP is used in
co-crystallization experiments, we developed a novel RNA affinity column that
purifies IRP and removes protein that is unable to bind IREs. Allerson CR, Loyevsky M, Mompoint F, Yikilmaz E, Altschul
SF, Madden T, Wootton JC., Kurantsin-Mills J, Kassim OO, Gordeuk VR, Rouault
TA. Expression of a recombinant IRP-like Plasmodium falciparum protein that
specifically binds putative plasmodial IREs. Mol Biochem Parasitol 2003;126:231-238. COLLABORATORS Victor Gordeuk, MD, Wolff
Kirsch, MD, Loma Linda University, Loma
Linda, CA Alan P. Koretsky, PhD, Laboratory of Functional and Molecular
Imaging, NINDS, Rodney L. Levine, MD, PhD, Laboratory of Biochemistry, NHLBI, Sriram Subramanium, PhD, Laboratory of Cell Biology, NCI, For more information, contact rouaultt@mail.nih.gov |