Previously, we identified and characterized the major iron metabolism–regulatory system in mammalian cells, determining that post-transcriptional mechanisms mediate iron–dependent alterations in expression of iron metabolism genes such as ferritin and the transferrin receptor. 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 (1) the inhibition of translation of ferritin mRNA and other transcripts with IREs near the 5´end and (2) the 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, the citric acid cycle enzyme. It functions as a cytosolic aconitase in cells that are iron-replete. Regulation of RNA-binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound to one that loses both iron and aconitase activity. 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 NifU genes implicated in bacterial iron-sulfur cluster assembly. We have shown that the products of these genes 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 abundant in mitochondria and cytosol and 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 Friedreich’s ataxia, iron-sulfur cluster biogenesis is impaired, and our studies aim to elucidate the role of frataxin in mammalian iron-sulfur cluster assembly. Our recent studies suggest that an iron-sulfur protein regulates mitochondrial iron homeostasis and that compromised iron-sulfur cluster assembly results in mitochondrial overload because of abnormal mitochondrial-to-nuclear signaling.
Rouault TA. Linking physiological functions of iron. Nat Chem Biol 2005 (in press).
Rouault TA, Tong WH. Opinion: Iron-sulphur cluster biogenesis and mitochondrial iron homeostasis. Nat Rev Mol Cell Biol 2005;6:345-351.
Iron-dependent degradation of IRP2: role of iron and heme
Ghosh
IRP2 also binds to IREs in iron-depleted cells, but, unlike IRP1, IRP2 is degraded in cells that are iron-replete. Experimental evidence indicates that IRP2 undergoes iron-catalyzed oxidation. The proteasome then selectively ubiquitinates and degrades the oxidized protein. 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 is 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 2003;31:247-255.
Rouault TA. Microbiology. Pathogenic bacteria prefer heme. Science 2004;305:1577-1578.
Rouault TA. The intestinal heme transporter revealed. Cell 2005;122:649-651.
Salvatore MF, Fisher B, Surgener SP, Gerhardt GA, Rouault T. Neurochemical investigations of dopamine neuronal systems in iron-regulatory protein 2 (IRP-2) knockout mice. Brain Res Mol Brain Res 2005;139:341-347.
Physiology and regulation of iron metabolism
Cooperman, Meyron-Holtz, Missirlis; in collaboration with Holmberg
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, we observed no abnormalities in iron metabolism associated with loss of IRP1 function. IRP2–/– mice developed a progressive movement disorder characterized by gait abnormalities and a Parkinsonian tremor. Animals accumulated ferritin iron in axons and developed 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 by magnetic resonance imaging. Vacuolar changes that develop as a result of neuronal cell body loss in regions such as the substantia nigra are detectable on histopathology and correlate with decreased T2 signals on MRI. However, 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 analyzing transgenic mice that overexpress ferritin subunits and transferrin receptor. To simplify the analysis of the role of iron metabolism in neurodegeneration, we developed a Drosophila model system and have 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 patients with Parkinson’s disease and related neurodegenerative diseases by sequencing candidate disease genes and using the new Clinical Center human MRI magnet. We also discovered that IRP2–/– mice have microcytic anemia and erythropoietic protoporphyria (EPP), and we have identified patients with unexplained EPP whom we can evaluate for IRP2 mutations.
Cooperman SS, Meyron-Holtz EG, Olivierre-Wilson H, Ghosh MC, McConnell JP, Rouault TA. Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood 2005;106:1084-1091.
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.
Meyron-Holtz EG, Ghosh MC, Rouault TA. Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo. Science 2004;306:2087-2090.
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, Tsai RYL, Berger UV, Sheng Z, 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 and high-resolution imaging
Yikilmaz
We have purified milligram quantities of Plasmodium falciparum IRP1 and IRP2 by overexpression in Pichia pastoris. 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. We have used overexpressed IRPs in biophysical studies and co-crystallization experiments.
Hodges M, Yikilmaz E, Patterson G, Kasvosve I, Rouault TA, Gordeuk VR, Loyevsky M. An iron regulatory-like protein expressed in Plasmodium falciparum displays aconitase activity. Mol Biochem Parasitol 2005;143:29-38.
Yikilmaz E, Rouault TA, Schuck P. Self-association and ligand-induced conformational changes of iron regulatory proteins 1 and 2. Biochemistry 2005;44:8470-8478.
Zhang P, Land W, Lee S, Juliani J, Lefman J, Smith SR, Germain D, Kessel M, Leapman R, Rouault TA, Subramaniam S. Electron tomography of degenerating neurons in mice with abnormal regulation of iron metabolism. J Struct Biol 2005;150:144-153.
Collaborators
Vineta Fellman, MD, PhD, Hospital for Children and Adolescents, Lund, Sweden
Victor Gordeuk, MD, Howard University Medical Center, Washington, DC
Sara Holmberg, BS, Georgetown University Medical Center, Washington, DC
Wolff Kirsch, MD, Loma Linda University, Loma Linda, CA
Alan P. Koretsky, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD
Rodney L. Levine, MD, PhD, Laboratory of Biochemistry, NHLBI, Bethesda, MD
Peter Schuck, PhD, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
Sriram Subramaniam, PhD, Laboratory of Cell Biology, NCI, Bethesda, MD
For further information, contact trou@helix.nih.gov.