Section on Human Iron Metabolism

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

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 Clinical Center human MRI magnet.

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, Martinez A, Yikilmaz E, Rouault TA. A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 over-expressed in Pichia Pastoris. RNA 2003;9:364-374.

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

Vineta Fellman, MD, PhD, Hospital for Children and Adolescents, Helsinki University Central Hospital, Finland

Victor Gordeuk, MD, Howard 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

Sriram Subramanium, PhD, Laboratory of Cell Biology, NCI, Bethesda, MD

For more information, contact rouaultt@mail.nih.gov