Regulation of Mammalian Intracellular Iron Metabolism and Biogenesis of Iron-Sulfur Proteins
- Tracey A. Rouault, MD, Head, Section on Human Iron Metabolism
- Wing Hang Tong, PhD, Staff Scientist
- Deliang Zhang, PhD, Staff Scientist
- Manik C. Ghosh, PhD, Research Assistant
- Anshika Jain, PhD, Visiting Fellow
- Gang Liu, PhD, Visiting Fellow
- Nunziata Maio, PhD, Visiting Fellow
- Samira Brooks, PhD, Postdoctoral Researcher, Division of Prevention and Epidemiology, NCI
- Anna Santamaria, PhD, Postdoctoral Fellow
- Hayden Ollivierre-Wilson, Animal Care Technician
- Anamika Singh, MS, Technical Contract Worker
- Gennadiy Kovtunovych, PhD, Special Volunteer
- Christina Porras, BS, PhD Candidate, Postbaccalaureate Fellow in the Brown NIH Joint Neurosciences Program
Our goal is to understand how mammals regulate intracellular and systemic iron metabolism to support processes that require iron and iron-sulfur clusters. Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) regulate the expression of numerous proteins involved in 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 the 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 the likely result of functional iron deficiency. Biochemically and with expression arrays, we studied, in IRP2–/– mice, the mechanisms that lead to anemia and neurodegeneration with motor neuron loss. We are using our mouse model of neurodegeneration to identify compounds that can prevent neurodegeneration; for example, we found that the antioxidant Tempol works by activating the latent IRE–binding activity of IRP1. Given that mitochondrial energy production is required to maintain axonal integrity and that motor neurons have the longest and most vulnerable axons, we hypothesize that mitochondrial dysfunction resulting from iron deficiency causes axonal degeneration. We discovered that deficiency in IRP1 causes polycythemia and pulmonary hypertension resulting from translational derepression of hypoxia-inducible factor (HIF) 2a through the IRE–IRP system. Our discovery introduces a new level of physiological regulation of erythropoiesis and provides a model for early pulmonary hypertension.
Our ongoing work on iron-sulfur cluster biogenesis has led to new insights into how mammalian iron-sulfur clusters are synthesized and transferred to appropriate recipient proteins. Several human diseases are now known to be caused by deficiencies in the iron-sulfur cluster biogenesis machinery. We developed a treatment for the rare disease ISCU (iron-sulfur cluster assembly enzyme) myopathy. By identifying a tripeptide motif common to many iron-sulfur recipient proteins, we developed an algorithm that facilitates discovery of previously unrecognized mammalian iron-sulfur proteins. Our work suggests that there are hundreds of previously unrecognized mammalian iron-sulfur proteins. Discovery of iron-sulfur cofactors will lead to breakthroughs in several research areas involving DNA repair, ribosomal biogenesis, mRNA translation, intermediary metabolism, and the regulation of the growth and energy-sensing pathways that are critical for determining the fates of many cell types.
The molecular basis for the regulation of intracellular iron metabolism in mammals
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 IRP1 and IRP2, two essential iron-sensing proteins. IRPs bind to IREs when iron levels are depleted, resulting in either inhibition of translation of ferritin mRNA and of 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, in iron-replete cells it 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 initially 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 in most cell types, but we discovered several cell types and accompanying phenotypes in which Irp2 expression cannot be sufficiently increased to compensate. 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; the treatment appears to work by recruiting the IRE–binding activity of IRP1. We found that motor neurons were the most adversely affected neurons in Irp2–/– mice and that neuronal degeneration accounted for the gait abnormalities. In collaboration with Grace Yoon, we discovered two IRP2–/– patients who suffered from severe neurodegenerative disease in infancy, and were bed-ridden or died as adolescents.
We discovered a form of the iron exporter ferroportin lacking the IRE at its 5′ end that is important in permitting iron to cross the duodenal mucosa in iron-deficient animals and in preventing developing erythroid cells from retaining high amounts of iron in iron-deficient animals. Our findings explain why microcytic anemia is usually the first physiological manifestation of iron deficiency in humans. Unexpectedly, we discovered that ferroportin is an abundant protein on mature red cells, where, as our work showed, it is needed to export free iron released from heme by oxidation. Using erythroid ferroportin knockout animals, we showed that absence of ferroportin results in accumulation of intracellular iron, increased oxidative stress, and reduced viability of cells in circulation.
Upon realizing that ferroportin is key to reducing free iron levels in red cells, we analyzed the Q248H mutation of ferroportin, which confers gain of function and reduces iron abundance in red cells. The Q248H mutation underwent positive selection in malarious regions of Africa, and we hypothesized that it conferred resistance to malaria by diminishing iron available to support growth of the malaria parasite in red cells. Upon infecting mice that lacked erythroid ferroportin with several malaria strains, we demonstrated that the mice experienced increased morbidity and mortality, likely because iron concentrations in red cells were high and supported parasite growth well. We noted that more than 8% of African Americans carry this allele, which has the potential to cause tissue iron overload in liver and kidney, perhaps accounting for some of the morbidities to which African Americans are unusually predisposed.
We recently discovered that loss of IRP1 causes polycythemia and pulmonary hypertension through derepression of hypoxia-inducible factor 2-alpha (HIF2a) translation in renal interstitial through the IRE–IRP system. We confirmed that overexpression of HIF2a drives production of erythropoietin and polycythemia in a mouse model of Chuvash polycythemia, and we discovered that we could reverse disease by activating Irp1 to repress HIF2a translation using TEMPOL, which converts Irp1 from the aconitase to the IRE–binding form. Phlebotomy has not been a very helpful therapy to the thousands of patients with Chuvash polycythemia is Russia, and we propose that oral Tempol supplementation could constitute a good therapeutic intervention. We also are conducting experiments with HIF2alpha inhibitors, which reveal that the drugs reverse polycythemia and pulmonary hypertension in our Irp1–/– and Chuvash polycythemia models.
We also elucidated the pathophysiology of intravascular hemolysis and hyposplenism in animals that lack heme oxygenase 1 (HMOX1). Their tissue macrophages die because they cannot metabolize heme after phagocytosis of red cells. To mitigate or reverse disease, we performed bone marrow transplants from wild-type animals to supply animals with functional macrophages; the bone marrow transplants were successful. We then discovered that the bone marrow transplant was not necessary by demonstrating that exogenously expanded wild-type macrophages can repopulate the reticuloendothelial system of Hmox1–/– mice, restore normal erythrophagocytosis, and reverse renal iron overload and anemia. Five human HMOX1–/– patients have been identified, but we believe this represents an underdiagnosed rare human disease that is often misdiagnosed.
Mammalian iron-sulfur cluster biogenesis
Our goal in studying mammalian iron-sulfur biogenesis is 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. We also seek to understand the role of iron-sulfur cluster assembly in the regulation of mitochondrial iron homeostasis and in 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 the 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 binds to RNA.
We identified numerous 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 we observed that mutations in several iron-sulfur cluster biogenesis proteins cause disease. Loss of frataxin, a protein that promotes the biosynthesis of heme and assembly and repair of iron-sulfur clusters by enhancing early steps of iron-sulfur cluster biogenesis, causes Friedreich's ataxia, which is characterized by progressive compromise of balance and cardiac function. In a cohort of patients of Swedish descent, we found that loss of the iron-sulfur cluster assembly enzyme ISCU causes skeletal myopathy. To explain the tissue specificity of the ISCU myopathy, we studied myoblasts and other patient-derived tissue samples and cell lines. We discovered that many factors contribute to insufficiency of ISCU in skeletal muscle, including more pronounced abnormal splicing and unusual sensitivity of ISCU to degradation upon exposure to oxidative stress. Thus, oxidative stress may impair the ability of tissues to repair damaged iron-sulfur clusters by directly damaging a key component of the biogenesis machinery. We discovered that antisense therapy would likely work as a treatment for ISCU myopathy patients, as we were able to correct the causal splicing defect in patient myoblasts using stable antisense RNAs that were manufactured by high-quality techniques suitable for use in patients. In one patient, we found that a splicing abnormality of glutaredoxin 5 was associated with sideroblastic anemia. In the affected tissues, mitochondrial iron overload is a feature common to all three diseases.
We identified a tripeptide motif, LYR, in apoproteins that are recipients of nascent iron-sulfur clusters. The cochaperone HSC20 binds to HSPA9, its partner HSP70–type chaperone, and the chaperone complex binds to ISCU bearing a nascent iron-sulfur cluster and to iron-sulfur cluster–recipient proteins. We identified several direct iron-sulfur–recipient proteins in a yeast two-hybrid assay, using HSC20 as bait. By studying one known iron-sulfur recipient, succinate dehydrogenase subunit B (SDHB), we discovered that several LYR motifs of the SDHB primary sequence engage the iron-sulfur transfer apparatus by binding to the C-terminus of HSC20, facilitating delivery of the three iron-sulfur clusters of SDHB. We further discovered that the assembly factor SDHAF1 also engages the iron-sulfur cluster transfer complex to facilitate transfer of iron-sulfur clusters to SDHB. The discovery of the LYR motif will aid in the identification of unknown iron-sulfur proteins, which are likely to be much more common in mammalian cells than had been previously appreciated. More recently, we discovered that, through recognition of LYR-like motifs in these recipient proteins, HSC20 is responsible for the delivery of iron-sulfur clusters to respiratory chain complexes I–II.
Using expression arrays, we analyzed 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.
Using informatics, over-expression of candidate proteins, and iron detection using ICP–MS (inductively coupled mass spectrometry), we identified many more iron-sulfur proteins that are involved in a wide range of metabolic pathways, ranging from intermediary metabolism, DNA repair, and RNA synthesis, and possibly regulation of cellular growth control. Iron-sulfur proteins will prove to be integral to the functioning and sensing of numerous pathways important in cellular functions.
We discovered that the mitochondrial protein ABCB7 (ATP-binding cassette sub-family B member 7) forms a complex with dimeric ferrochelatase, which binds ABCB10 to the other half of the ferrochelatase dimer. Our preliminary results suggest that ABCB7 may represent a mitochondrial heme exporter.
Additional Funding
- Bench-to-Bedside Award: Analysis of whether the Ferroportin Q248H mutation prevalent in Africans and African Americans predisposes to unrecognized pathological tissue iron overload and disease
Publications
- Kim KS, Zhang DL, Kovtunovych G, Ghosh MC, Ollivierre H, Eckhaus MA, Rouault TA. Infused wild-type macrophages reside and self-renew in the liver to rescue the hemolysis and anemia of Hmox1-deficient mice. Blood Adv 2018;2(20):2732-2743.
- Zhang DL, Wu J, Shah BN, Greutélaers KC, Ghosh MC, Ollivierre H, Su XZ, Thuma PE, Bedu-Addo G, Mockenhaupt FP, Gordeuk VR, Rouault TA. Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk. Science 2018;359(6383):520-1523.
- Kim KS, Maio N, Singh A, Rouault TA. Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAO1-mediated transfer to recipients. Hum Mol Genet 2018;27(5):837-852.
- Costain G, Ghosh MC, Maio N, Carnevale A, Si YC, Rouault TA, Yoon G et al. Absence of iron-responsive element-binding protein 2 causes a novel neurodegenerative syndrome. Brain 2019;142(5):1195-1202.
- Maio N, Kim KS, Holmes-Hampton G, Singh A, Rouault TA. Dimeric ferrochelatase bridges ABCB7 and ABCB10 homodimers in an architecturally defined molecular complex required for heme biosynthesis. Haematologica 2019;104(9):1756-1767.
Collaborators
- J. Martin Bollinger, PhD, Penn State University, University Park, PA
- Victor Gordeuk, MD, University of Illinois College of Medicine, Chicago, IL
- Carsten Krebs, PhD, Penn State University, University Park, PA
- W. Marston Linehan, MD, Urologic Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD
- John F. Tisdale, MD, Molecular and Clinical Hematology Branch, NIDDK, Bethesda, MD
- Grace Yoon, MD, The Hospital for Sick Children, Toronto, Canada
Contact
For more information, email trou@helix.nih.gov or visit https://irp.nih.gov/pi/tracey-rouault.
