REGULATION OF INTRACELLULAR IRON METABOLISM
, Head, Section on Human Iron Metabolism
Wing Hang Tong, PhD, Staff Scientist
Manik C. Ghosh, PhD, Research Chemist
Sharon Cooperman, MD, Senior Fellow
Kuanyu Li, PhD, Visiting Fellow
Fanis Missirlis, PhD, Visiting Fellow
Yanbo Shi, PhD, Visiting Fellow
Helge Uhrigshardt, PhD, Visiting Fellow
Hong Ye, PhD, Visiting Fellow
Deliang Zhang, PhD, Visiting Fellow
Anamika Singh, BS, Technical Trainee
Hayden Ollivierre-Wilson, Animal CareTechnician
Robert Hughes, College Student
Our overall goal is to understand how mammals regulate iron metabolism. In cells, iron-regulatory proteins 1 and 2 (IRP1 and IRP2) regulate expression of numerous proteins of iron metabolism. In cells that are iron-depleted, the proteins bind to RNA stem-loops in transcripts known as iron-responsive elements (IREs). IRP binding stabilizes the mRNA that encodes the transferrin receptor and represses translation of transcripts that contain IREs near the 5¢ end of ferritin H and L chains. IRP1 is an iron-sulfur protein that functions as an aconitase in iron-replete cells. IRP2 is homologous to IRP1, but it undergoes iron-dependent degradation in iron-replete cells. In mouse models, loss of IRP2 results in mild anemia, erythropoietic protoporphyria, and adult-onset neurodegeneration. These phenotypes are likely the result of functional iron deficiency. Using a variety of biochemical and expression arrays, we are studying the mechanisms that lead to anemia and neurodegeneration in IRP2−/− mice. In addition, we are using our mouse model of neurodegeneration to identify compounds that can prevent neurodegeneration and have found one that appears to work. We are evaluating the possibility that loss of IRP2 in humans may cause erythropoietic protoporphyria, adult-onset neurodegeneration, and some mild anemias.
The molecular basis for regulation of intracellular iron metabolism
In previous years, our laboratory identified and characterized the cis and trans elements mediating the iron-dependent alterations in the abundance of ferritin and the transferrin receptor. 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, IRP1 and IRP2. IRPs bind to IREs when iron levels are depleted, resulting in either inhibition of translation of ferritin mRNA and other transcripts that contain an IRE in the 5¢ untranslated regions (UTR) or stabilization of the transferrin receptor mRNA and possibly other transcripts that contain IREs in the 3¢UTR. The IRE-binding activity of IRP1 depends on the presence of an iron-sulfur cluster (see “Mammalian iron-sulfur cluster biogenesis” below). IRP2 also binds to IREs in iron-depleted cells, but, unlike IRP1, is degraded in iron-replete cells. Experimental evidence indicates that IRP2 binds to iron and undergoes iron-catalyzed oxidation. In iron-replete cells, IRP2 is selectively ubiquitinated and degraded by the proteasome. To approach questions about 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 develop a progressive neurologic syndrome characterized by gait abnormalities and axonal degeneration. Ferritin overexpression occurs in affected neurons and in protrusions of oligodendrocytes into the space created by axonal degeneration. IRP2−/− animals develop iron-insufficiency anemia and erythropoietic protoporphyria. In animals that lack IRP1, IRP 2 compensates for loss of IRP1 regulatory activity. Animals that lack both IRP1 and IRP2 die as early embryos. The adult-onset neurodegeneration of adult IRP2−/− mice is exacerbated when one copy of IRP1 is also deleted. IRP2−/− mice offer a unique example of spontaneous adult-onset, slowly progressive neurodegeneration; analyses of gene expression and iron status at various stages of disease are ongoing. At the same time, small-molecule treatments to prevent neurodegeneration have yielded promising results.
Lind MI, Missirlis F, Melefors O, Uhrigshardt H, Kirby K, Phillips JP, Söderhäll K, Rouault TA. Of two cytosolic aconitases expressed in Drosophila, only one functions as an iron-regulatory protein. J Biol Chem 2006;281:18707-14.
Rouault TA. Biochemistry if the RNA fits, use it. Science 2006;314:1886-7.
Rouault TA, Cooperman S. Brain iron metabolism. Semin Pediatr Neurol 2006;13:142-8.
Smith SR, Ghosh MC, Ollivierre-Wilson H, Hang Tong W, Rouault TA. Complete loss of iron regulatory proteins 1 and 2 prevents viability of murine zygotes beyond the blastocyst stage of embryonic development. Blood Cells Mol Dis 2006;36:283-7.
Zhang D, Meyron-Holtz E, Rouault TA. Renal iron metabolism: transferrin iron delivery and the role of iron regulatory proteins. J Am Soc Nephrol 2007;18:401-6.
Mammalian iron-sulfur cluster biogenesis
Our goals in studying mammalian iron-sulfur biogenesis are to understand how iron-sulfur prosthetic groups are assembled and delivered to target proteins in the various compartments of mammalian cells, including mitochondria, cytosol, and nucleus. In addition, we seek to understand the role of iron-sulfur cluster assembly in the regulation of mitochondrial iron homeostasis as well as the pathogenesis of diseases such as Friedreich ataxia and sideroblastic anemia, which are both characterized by incorrect regulation of mitochondrial iron homeostasis.
IRP1 is an iron-sulfur protein related to mitochondrial aconitase, which is a citric acid cycle enzyme that functions as a cytosolic aconitase in iron-replete cells. Regulation of 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. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA-binding protein in iron-depleted cells is an apoprotein. 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 those encoded by NifS, ISCU, and Nif U genes implicated in bacterial iron-sulfur cluster assembly and showed that these gene products facilitate assembly of the iron- sulfur cluster of IRP1.
Babady NE, Carelle N, Wells RD, Rouault TA, Hirano M, Lynch DR, Delatycki MB, Wilson RB, Isaya G, Puccio H. Advancements in the pathophysiology of Friedreichs Ataxia and new prospects for treatments. Mol Genet Metab 2007;92:23-35.
Li K, Tong WH, Hughes RM, Rouault TA. Roles of the mammalian cytosolic cysteine desulfurase, ISCS, and scaffold protein, ISCU, in iron-sulfur cluster assembly. J Biol Chem 2006;281:12344-51.
Rouault TA. The role of iron regulatory proteins in mammalian iron homeostasis and disease. Nat Chem Biol 2006;2:406-14.
Tong WH, Rouault TA. Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab 2006;3:199-210.
Tong WH, Rouault TA. Metabolic regulation of citrate and iron by aconitases: role of iron-sulfur cluster biogenesis. Biometals 2007;20:549-64.
COLLABORATORS
Clara Camaschella, MD, Università Vita-Salute San Raffaele, Milan, Italy
Kenneth H. Fischbeck, MD, Neurogenetics Branch, NINDS, Bethesda, MD
Ronald Haller, MD, University of Texas Southwestern Medical Center, Dallas, TX
Richard Holm, PhD, Harvard University, Cambridge, MA
Boi Hanh Huynh, PhD, Emory University, Atlanta, GA
Rodney L. Levine, MD, PhD, Laboratory of Biochemistry, NHLBI, Bethesda, MD
Andrew B. Singleton, PhD, Laboratory of Neurogenetics, NIA, Bethesda, MD
For further information, contact rouault@mail.nih.gov.
