This project aims to understand the molecular basis for regulation of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. 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 the inhibition of translation of ferritin mRNA and other transcripts that contain an IRE in the 5 untranslated regions, or in stabilization of the transferrin receptor mRNA and possibly other transcripts that contain IREs in the 3UTR. The IRE-binding activity of IRP1 depends on whether the protein contains an iron-sulfur cluster (see project HD008814-01). IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are iron- replete. In iron-replete cells, IRP2 is selectively ubiquitinated by FBXL5 and degraded by the proteasome. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, there are subtle 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 over-expression 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, and analyses of gene expression and iron status at various stages of disease are ongoing. We have found that lower motor neurons are very adversely affected, developing axonopathy and death. In addition, small molecule treatment with the stable nitroxide, Tempol, prevents neurodegeneration in IRP2-/- animals. We characterized metabolism in an HLRCC cell line and discovered that AMPK is down, which leads to reduced p53 and DMT1, an iron importer. The iron deficiency that arises as a consequence alters aerobic glycolysis. Only HIF1 alpha is significantly elevates, whereas HIF2 alpha expression is repressed by IRP activation. These metabolic changes lead to high storage of glycogen and fatty acids, which enables these cancer cells to store large amounts of energy that may fuel them during when they metastasize and temporarily lose access to nutrients.

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Zhang, De-Liang; Rouault, Tracey A (2018) How does hepcidin hinder ferroportin activity? Blood 131:840-842
Zhang, De-Liang; Wu, Jian; Shah, Binal N et al. (2018) Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk. Science 359:1520-1523
Ghosh, Manik C; Zhang, De-Liang; Ollivierre, Hayden et al. (2018) Translational repression of HIF2? expression in mice with Chuvash polycythemia reverses polycythemia. J Clin Invest 128:1317-1325
Rouault, Tracey A; Maio, Nunziata (2017) Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways. J Biol Chem 292:12744-12753
Maio, Nunziata; Kim, Ki Soon; Singh, Anamika et al. (2017) A Single Adaptable Cochaperone-Scaffold Complex Delivers Nascent Iron-Sulfur Clusters to Mammalian Respiratory Chain Complexes I-III. Cell Metab 25:945-953.e6
Rouault, Tracey A (2016) Mitochondrial iron overload: causes and consequences. Curr Opin Genet Dev 38:31-37
Cloonan, Suzanne M; Glass, Kimberly; Laucho-Contreras, Maria E et al. (2016) Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice. Nat Med 22:163-74
Maio, Nunziata; Ghezzi, Daniele; Verrigni, Daniela et al. (2016) Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB. Cell Metab 23:292-302
Maio, N; Rouault, T A (2016) Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20. Metallomics 8:1032-1046
Holmes-Hampton, Gregory P; Crooks, Daniel R; Haller, Ronald G et al. (2016) Use of antisense oligonucleotides to correct the splicing error in ISCU myopathy patient cell lines. Hum Mol Genet 25:5178-5187

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