Tracey A. Rouault, MD, Head, Section on Human Iron Metabolism
Sharon Cooperman, MD, Senior Fellow
Manik Ghosh, PhD, Chemist
Wing Hang Tong, PhD, Staff Scientist
Kuanyu Li, PhD, Postdoctoral Fellow
Fanis Missirlis, PhD, Postdoctoral Fellow
Helge Uhrigshardt, PhD, Postdoctoral Fellow
Emine Yikilmaz, PhD, Postdoctoral Fellow
Yanbo Shi, PhD, Visiting Fellow
Hong Ye, PhD, Visiting Fellow
Deliang Zhang, PhD, Visiting Fellow
Hayden Olivierre-Wilson, Contractor (Animal Care)

Previously, our laboratory identified and characterized the major iron metabolism--regulatory system in mammalian cells. 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 in the extension of the half-life of the transferrin receptor mRNA and possibly of 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, Ye, Shi

Related to mitochondrial aconitase, a citric acid cycle enzyme, IRP1 is an iron-sulfur protein that 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 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 identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and NifU genes implicated in bacterial iron-sulfur cluster assembly; specific isoforms of these gene products facilitate assembly of the iron-sulfur cluster of IRP1 in the cytosol. 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. In the human disease 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.

Iron-dependent degradation of IRP2: role of iron and heme

Ghosh

IRP2 also binds to IREs in iron-depleted cells but, unlike IRP1, it 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 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 if heme is a cofactor for a trans-acting factor involved in iron-dependent degradation.

Physiology and regulation of iron metabolism

Cooperman, Missirlis, Zhang, Ghosh

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 develop a progressive movement disorder characterized by gait abnormalities and a Parkinsonian tremor. Animals accumulate ferritin iron in axons, which degenerate. These findings are greatly accentuated in animals lacking 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. Animals lacking 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 transgenic mice that overexpress ferritin subunits and transferrin receptor and analyzed them. We discovered that IRP2-/- mice have a microcytic anemia and erythropoietic protoporphyria (EPP), and we have identified patients with unexplained EPP whom we can evaluate for IRP2 mutations. Accordingly, we plan to study selected humans with Parkinson's disease and related neurodegenerative diseases by sequencing candidate disease genes and using the new Clinical Center's human MRI magnet.

Regulation of iron metabolism in Drosophila

Missirlis

To analyze more simply the role of iron metabolism in neurodegeneration, 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.

Structural characterization of IRPs and IREs and high-resolution imaging

Yikilmaz

We have purified milligram quantities of IRP1 and IRP2 by overexpression in Pichia pastoris from Plasmodium falciparum. To ensure high-quality IRP for co-crystallization experiments, we developed a novel RNA affinity column that purifies IRP and removes protein that is unable to bind to IREs. We have used overexpressed IRPs in biophysical studies and co-crystallization experiments.

COLLABORATORS

Vineta Fellman, MD, PhD, Hospital for Children and Adolescents, Lund, Sweden
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
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.