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REGULATION OF INTRACELLULAR IRON METABOLISM
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Tracey A. Rouault, MD, Head, Section on Human Iron Metabolism Manik Ghosh, PhD, Senior Fellow |
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| Previously, our laboratory identified and characterized the cis and trans elements that regulate iron-dependent alterations in the expression of 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 cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2). IRPs bind 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 prolongation of the half-life of the transferrin receptor mRNA. Iron-sulfur cluster assembly Tong, Brazzolotto IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme that functions as a cytosolic aconitase in cells that are iron-replete. Regulation of the RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound to a form 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 have identified mammalian enzymes of the iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly and have shown that these gene products facilitate assembly of the iron-sulfur cluster of IRP1. We have 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, a protein that assembles a [4Fe-4S] cluster, is an abundant protein in mitochondria and the cytosol. NFU may function as a scaffold for iron-sulfur cluster assembly by donating its newly assembled clusters to recipient proteins. 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, IRP2 is degraded in cells that are iron-replete. 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 numerous other proteins are degraded by a pathway in which oxidative modification is 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 if 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. In: Blood Cells, Molecules and Disease; in press. Drake SK, Bourdon E, Wehr N, Levine RL, Backlund P, Yergey A, Rouault TA. Numerous proteins in mammalian cells are prone to iron-dependent oxidation and proteasomal degradation. Dev Neurosci 2002;24:114-124. 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 Smith, Cooperman, Grabill, Jui-chen, Meyron-Holtz, Land, Missirlis To approach questions about the physiology of iron metabolism, we have 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 develop a progressive movement disorder characterized by gait abnormalities and tremor. Animals accumulate iron in axons and develop axonal degeneration. Ferritin overexpression occurs in affected neurons, and ferritin accumulation occurs in axons. These findings are greatly accentuated in animals that lack one copy of IRP1 in addition to both copies of IRP2. IRP2 is the predominant regulator of posttranscriptional 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 can be detected on MRI and pathology. Animals that lack both IRP1 and IRP2 do not survive past the blastocyst stage. neuronal pathology in iron regulatory protein-2 knockout mice. Brain Res 2003;971:95-106. 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;278;47365-47369. Structural characterization of IRPs and IREs Yikilmaz We have purified milligram quantities of IRP1 and IRP2 and are working on crystallization of each IRP. In addition, we are trying to cocrystallize each IRP in a complex with IRE. We have characterized and overexpressed an IRP-like protein from Plasmodium falciparum. To ensure that high-quality IRP is used in cocrystallization experiments, we developed a novel RNA affinity column that purifies IRP and removes protein that is unable to bind to 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 Greg Gearhardt, PhD, University of Kentucky, Lexington KY Victor Gordeuk, MD, Howard University Medical Center, Washington DC Wolff Kirsch, MD, Loma Linda University, CA Alan P. Koretsky, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda MD Sriram Subramaniam, PhD, Laboratory of Cell Biology, NCI, Bethesda MD Alfred Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda MD For further information, contact trou@helix.nih.gov |
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