We aim to understand how mammals regulate intracellular and systemic iron metabolism. Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) regulate the 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, which is characterized by limb weakness and might be diagnosed as amyotrophic lateral sclerosis in humans.
In previous years, our laboratory identified and characterized the cis and trans elements mediating 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 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. 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, IRP2 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. We discovered that motor neurons were the most adversely affected neurons in IRP2−/− mice, and neuronal degeneration accounted for the gait abnormalities. We discovered a form of the iron exporter, ferroportin, that lacks an IRE at its 5′ end and is important in permitting iron to cross the duodenal mucosa in iron-deficient animals and in preventing developing erythroid cells from consuming excessive amounts of iron in iron-deficient animals. We concluded that, in animals that lack heme oxygenase, the main cause of tissue iron distribution was loss of viable tissue macrophages in the spleen and liver because of the cells' inability to survive after phagocytosis of red cells.
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, the cytosol, and the 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, which are implicated in bacterial iron-sulfur cluster assembly, and showed that the gene products facilitate assembly of the iron-sulfur cluster of IRP1. Mutations in several iron-sulfur cluster biogenesis proteins cause disease. Loss of frataxin causes Friedreich's 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. In collaborative work, we discovered that mutations of two new iron sulfur cluster assembly proteins, NFU and BOLA3, which are needed to provide iron sulfur clusters to lipoate-dependent enzymes. Using expression arrays, we are analyzing the mechanisms by which compromised 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.
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