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Regulation of Intracellular Iron Metabolism

Tracey A. Rouault, MD, Head
  • Tracey A. Rouault, MD, Head, Section on Human Iron Metabolism
  • Dan Crooks, MS, Graduate Student, Georgetown NIH Partnership Program
  • Manik C. Ghosh, PhD, Research Assistant
  • Suh-Yeong Jeong, PhD, Visiting Fellow
  • Gennadiy Kovtunovych, PhD, Visiting Fellow
  • Kuanyu Li, PhD, Visiting Fellow
  • Hayden Ollivierre-Wilson, Animal Care Technician
  • Neetu Saxena, PhD, Visiting Fellow
  • Thomas Senecal, Eleanor Roosevelt High School Student
  • Yanbo Shi, PhD, Visiting Fellow
  • Anamika Singh, MS, Technical Trainee
  • Wing Hang Tong, PhD, Staff Scientist
  • Helge Uhrigshardt, PhD, Visiting Fellow
  • Hong Ye, PhD, Visiting Fellow
  • Deliang Zhang, PhD, Visiting Fellow

We aim to understand how mammals regulate intracellular and systemic iron metabolism. Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) regulate expression of numerous proteins of iron metabolism. In iron-depleted cells, the proteins bind to RNA stem-loops in transcripts known as iron-responsive elements (IRE). IRP binding stabilizes the mRNA that encodes the transferrin receptor and represses the 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 undergoes iron-dependent degradation in iron-replete cells. In mouse models, loss of IRP2 results in mild anemia, erythropoietic protoporphyria, and adult-onset neurodegeneration—all likely the result of functional iron deficiency. Biochemically and with expression arrays, we study the mechanisms that lead to anemia and neurodegeneration in IRP2−/− mice and are using our mouse model of neurodegeneration to identify compounds that can prevent neurodegeneration; we found that the antioxidant Tempol works by activating the latent IRE-binding activity of IRP1. We are evaluating the possibility that loss of IRP2 in humans may cause mild refractory anemia and adult-onset neurodegeneration characterized by limb weakness, which might be diagnosed as amyotrophic lateral sclerosis in humans.

The molecular basis for regulation of intracellular iron metabolism in mammals

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 (1) inhibition of translation of ferritin mRNA and other transcripts that contain an IRE in the 5′-untranslated regions (UTR) or (2) 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 then 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’s 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. Dietary supplementation with the stable nitroxide Tempol prevents neurodegeneration, and this treatment appears to work by recruiting IRE-binding activity of IRP1. 

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 and the pathogenesis of diseases such as Friedreich’s 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 reveal 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 the NifS, ISCU, and NifU genes implicated in bacterial iron-sulfur cluster assembly and showed that the gene products facilitate assembly of the iron-sulfur cluster of IRP1. Mutations of several iron-sulfur cluster biogenesis proteins cause disease. Loss of frataxin causes Friedreich ataxia, which is characterized by progressive compromise of balance and cardiac function. Loss of ISCU causes skeletal myopathy in patients of Swedish descent, and a splicing abnormality of glutaredoxin 5 was found to be associated with a sideroblastic anemia in one patient. In the affected tissues, mitochondrial iron overload is a feature common to all three diseases. Using expression arrays, we are analyzing the mechanisms by which compromise of mitochondrial iron-sulfur cluster biogenesis leads to mitochondrial iron overload. We postulate that regulation of mitochondrial iron homeostasis depends on intact synthesis of an iron-sulfur cluster–regulatory protein. Once this pathway is better understood, insights may lead to treatments for several rare diseases.

Additional Funding

  • Friedreich Ataxia Research Association

Publications

  • Ghosh MC, Tong WH, Zhang D, Ollivierre-Wilson H, Singh A, Krishna MC, Mitchell JB, Rouault TA. Tempol mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice. Proc Natl Acad Sci USA 2008 105:12028-12033.
  • Li K, Besse EK, Ha D, Kovtunovych G, Rouault TA. Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia. Hum Mol Genet 2008 17:2265-2273.
  • Rouault TA, Tong WH. Iron-sulfur cluster biogenesis and human disease. Trends Genet 2008 24:398-407.
  • Shi Y, Ghosh MC, Tong WH, Rouault TA. Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis. Hum Mol Genet 2009 18:3014–3025.
  • Zhang DL, Hughes RM, Ollivierre-Wilson H, Ghosh MC, Rouault TA. A ferroportin transcript that lacks an ironresponsive element enables duodenal and erythroid precursor cells to evade translational repression. Cell Metab 2009 9:461-473.

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, PhD, Laboratory of Biochemistry, NHLBI, Bethesda, MD
  • Andrew B. Singleton, PhD, Laboratory of Neurogenetics, NIA, Bethesda, MD

Contact

For more information, email trou@helix.nih.gov or visit https://irp.nih.gov/pi/tracey-rouault.

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