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Publisher Summary Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression of proteins implicated in iron and energy metabolism. IRE/IRP1 interactions modulate mRNA translation or stability and result in homeostatic... more
Publisher Summary Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression of proteins implicated in iron and energy metabolism. IRE/IRP1 interactions modulate mRNA translation or stability and result in homeostatic adaptations to changes in iron availability. IRP1 belongs to the family of iron–sulfur isomerases. Its genetic activity is regulated by the iron dependent assembly–disassembly of a cubane, aconitase-type [4Fe–4S] cluster. Direct administration of hydrogen peroxide to cells leads to a rapid activation of IRP1 to its IRE-binding form—IRPI activation. This chapter describes the basic methods that have been developed and applied to study the activation of IRPI by hydrogen peroxide. These include the electrophoretic mobility shift assay to detect IRE-binding activity, the chemiluminescence luminol/hypochlorite assay to detect extracellular hydrogen peroxide, the method for enzymatic generation of hydrogen peroxide at steady-state levels, and the fluorometric assay to monitor relative intracellular hydrogen peroxide levels. In addition, the chapter describes key experiments that have provided insights regarding the mechanism and the physiological implications of IRP1 activation by hydrogen peroxide in cultured B6 fibroblasts, in permeabilized B6 fibroblasts, and in the intact rat liver.
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Hepcidin is a liver‐derived peptide hormone that limits iron egress from tissues to the bloodstream. It operates by binding to the iron exporter ferroportin, which blocks iron transport and tags ferroportin for degradation. Genetic... more
Hepcidin is a liver‐derived peptide hormone that limits iron egress from tissues to the bloodstream. It operates by binding to the iron exporter ferroportin, which blocks iron transport and tags ferroportin for degradation. Genetic hepcidin inactivation leads to hereditary hemochromatosis, a disease of iron overload. We used wild‐type and Hjv‐/‐ mice, a model of hemochromatosis, to examine the expression of ferroportin and other proteins of iron metabolism in hepcidin target tissues. The animals were previously subjected to dietary iron manipulations. In Hjv‐/‐ mice, hepcidin messenger RNA correlated significantly with hepatic iron load (r = 0.8211, P < 0.001), but was substantially lower compared with wild‐type controls. Duodenal ferroportin and divalent metal transporter 1 (DMT1), as well as splenic and hepatic ferroportin, were overexpressed in these animals. A high‐iron diet (2% carbonyl iron) suppressed duodenal DMT1 levels in both wild‐type and Hjv‐/‐ mice; however, it did not affect duodenal ferroportin expression in Hjv‐/‐ mice, and only reduced it in wild‐type mice. In contrast, the high‐iron diet decreased splenic ferroportin exclusively in Hjv‐/‐ mice, whereas it induced hepatic ferroportin exclusively in wild‐type mice. Conclusion: Our data show that dietary iron differentially affects ferroportin expression in mouse tissues and are consistent with hepcidin‐dependent and hepcidin‐independent mechanisms for ferroportin regulation. In the Hjv‐/‐ mouse model of hemochromatosis, duodenal ferroportin remains unresponsive to iron but DMT1 is appropriately iron‐regulated.
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Despite advances in our knowledge and attempts to improve therapies, β-thalassemia remains a prevalent disorder with increased risk for the development of cardiomyopathy. Using an untargeted discovery-based lipidomic workflow, we... more
Despite advances in our knowledge and attempts to improve therapies, β-thalassemia remains a prevalent disorder with increased risk for the development of cardiomyopathy. Using an untargeted discovery-based lipidomic workflow, we uncovered that transfusion-dependent thalassemia (TDT) patients had a unique circulating lipidomic signature consisting of 387 lipid features, allowing their significant discrimination from healthy controls (Q-value < 0.01). In particular, TDT patients had elevated triacylglycerols and long-chain acylcarnitines, albeit lower ether phospholipids or plasmalogens, sphingomyelins, and cholesterol esters, reminiscent of that previously characterized in cardiometabolic diseases resulting from mitochondrial and peroxisomal dysfunction. Discriminating lipid (sub)classes correlated differentially with clinical parameters, reflecting blood (ether phospholipids) and iron (cholesterol ester) status or heart function (triacylglycerols). We also tested 15 potential se...
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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ABSTRACT Iron is an essential nutrient but also a potential biohazard. Elaborate homeostatic mechanisms have evolved to regulate dietary iron absorption at levels sufficient to satisfy metabolic needs and prevent the accumulation of metal... more
ABSTRACT Iron is an essential nutrient but also a potential biohazard. Elaborate homeostatic mechanisms have evolved to regulate dietary iron absorption at levels sufficient to satisfy metabolic needs and prevent the accumulation of metal excess. Internalized dietary iron enters the pool of plasma transferrin for delivery into the erythron and other tissues. Nevertheless, in healthy adults, the daily contribution of dietary iron for erythropoiesis is minimal and the vast majority of circulating transferrin-iron derives from macrophages, that eliminate senescent red blood cells and recycle their iron. Cellular iron uptake is mediated by endocytosis of iron-loaded transferrin upon binding to its transferrin receptor 1 (TfR1). Excess of intracellular iron that is not required for metabolic purposes is stored within ferritin. The expression of TfR1 and ferritin is coordinately and reciprocally controlled by a post-transcriptional mechanism. This involves two cytoplasmic iron regulatory proteins (IRP1 and IRP2), which interact with the iron responsive elements (IREs) of TfR1 and ferritin mRNAs. IRE/IRP interactions that occur in iron-deficient cells, stabilize TfR1 mRNA and inhibit ferritin mRNA translation. In iron-replete cells, IRP1 assembles an aconitase-type [4Fe-4S] 2+ cluster, which precludes IRE-binding. By contrast, IRP2 undergoes iron-dependent proteasomal degradation following ubiquitination. IRPs control the expression of additional mRNAs and respond not only to cellular iron levels but also to other stimuli, such as oxygen, oxidative stress and nitric oxide. The targeted disruption of both IRP1 and IRP2 in mice is associated with early embryonic lethality, underlying the physiological significance of the IRE/IRP regulatory system. While the ablation of IRP1 alone does not manifest any discernible pathology, IRP2(-/-) mice exhibit microcytic anemia and neurological defects. The ongoing development of mouse strains with spatial and temporal disruption of IRPs is providing further insight on their physiological functions.
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Research Interests: Hypoxia, Oxidative Stress, Cell line, Humans, Proteasome, and 14 moreMice, Animals, Siderophores, Iron, Hemagglutinin, Hydrogen Peroxide, Oxygen, Quaternary Ammonium Compounds, DNA binding proteins, Oxidation-Reduction, Ferric Compounds, Oxidants, Response Elements, and Biochemistry and cell biology
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Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a... more
Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages. The binding of iron-laden transferrin to the cell-surface transferrin receptor 1 results in endocytosis and uptake of the metal cargo. Internalized iron is transported to mitochondria for the synthesis of haem or iron–sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. Iron metabolism is controlled at different levels and by diverse mechanisms. The present review summarizes basic concepts of iron transport, use and storage and focuses on the IRE (iron-responsive element)/IRP (iron-regulatory protein) system, a well known post-transcriptional regula...
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Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron... more
Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells wi...
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Key Points TfrcAlb-Cre mice exhibit low hepatocellular iron and develop mild hypoferremia and microcytosis due to inappropriate hepcidin expression. Tfr1 is essential for fine-tuning hepcidin responses to hepatocellular iron load but is... more
Key Points TfrcAlb-Cre mice exhibit low hepatocellular iron and develop mild hypoferremia and microcytosis due to inappropriate hepcidin expression. Tfr1 is essential for fine-tuning hepcidin responses to hepatocellular iron load but is dispensable for basal iron supply to hepatocytes.
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Hepcidin is a key hormonal regulator of systemic iron homeostasis and its expression is induced by iron or inflammatory stimuli. Genetic defects in iron signaling to hepcidin lead to “hepcidinopathies” ranging from hereditary... more
Hepcidin is a key hormonal regulator of systemic iron homeostasis and its expression is induced by iron or inflammatory stimuli. Genetic defects in iron signaling to hepcidin lead to “hepcidinopathies” ranging from hereditary hemochromatosis to iron-refractory iron deficiency anemia, which are disorders caused by hepcidin deficiency or excess, respectively. Moreover, dysregulation of hepcidin is a pathogenic cofactor in iron-loading anemias with ineffective erythropoiesis and in anemia of inflammation. Experiments with preclinical animal models provided evidence that restoration of appropriate hepcidin levels can be used for the treatment of these conditions. This fueled the rapidly growing field of hepcidin therapeutics. Several hepcidin agonists and antagonists, as well as inducers and inhibitors of hepcidin expression have been identified to date. Some of them were further developed and are currently being evaluated in clinical trials. This review summarizes the state of the art.
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Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis... more
Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoiesis. In β-thalassemia, β-globin synthesis is reduced causing α-globin accumulation. Unpaired globin chains, with heme attached to them, accumulate in thalassemic erythroblasts causing oxidative stress and the premature cell death. We hypothesize that in β-thalassemia heme oxygenase (HO) 1 could play a pathogenic role in the development of anemia and ineffective erythropoiesis. To test this hypothesis, we exploited a mouse model of β-thalassemia intermedia, Th3/We observed that HO inhibition using tin protoporphyrin IX (SnPP) decreased heme-iron recycling in the liver and ameliorated anemia in the Th3/mice. SnPP administration led to a decre...
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Cellular iron homeostasis is accomplished by the coordinated regulated expression of the transferrin receptor and ferritin, which mediate iron uptake and storage, respectively. The mechanism is posttranscriptional and involves two... more
Cellular iron homeostasis is accomplished by the coordinated regulated expression of the transferrin receptor and ferritin, which mediate iron uptake and storage, respectively. The mechanism is posttranscriptional and involves two cytoplasmic iron regulatory proteins, IRP1 and IRP2. Under conditions of iron starvation, IRPs stabilize the transferrin receptor and inhibit the translation of ferritin mRNAs by binding to &amp;amp;amp;amp;amp;amp;amp;amp;quot;iron responsive elements&amp;amp;amp;amp;amp;amp;amp;amp;quot; (IREs) within their untranslated regions. The IRE/IRP system also controls the expression of additional IRE-containing mRNAs, encoding proteins of iron and energy metabolism. The activities of IRP1 and IRP2 are regulated by distinct posttranslational mechanisms in response to cellular iron levels. Thus, in iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster, which prevents IRE binding, while IRP2 undergoes proteasomal degradation. IRP1 and IRP2 also respond, albeit differentially, to iron-independent signals, such as hydrogen peroxide, hypoxia, or nitric oxide. Basic principles of the IRE/IRP system and recent advances in understanding the regulation and the function of IRP1 and IRP2 are discussed.
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Non-transferrin-bound iron (NTBI) emerges in plasma of patients with systemic iron overload, but has also been documented in further pathological conditions. Quantification of NTBI can be useful for diagnosis and management of these... more
Non-transferrin-bound iron (NTBI) emerges in plasma of patients with systemic iron overload, but has also been documented in further pathological conditions. Quantification of NTBI can be useful for diagnosis and management of these disorders. However, currently available detection methods are tedious and often inaccurate, hampering wide applicability. In this issue of the Biochemical Journal, Ma et al. report the development of a novel assay for NTBI measurement, based on an iron-sensitive fluorescent probe that is linked to magnetic beads. The approach offers several advantages over existing technology and may bring NTBI assessment closer to the clinic.
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Iron is essential for energy metabolism, and states of iron deficiency or excess are detrimental for organisms and cells. Therefore, iron and carbohydrate metabolism are tightly regulated. Serum iron and glucose levels are subjected to... more
Iron is essential for energy metabolism, and states of iron deficiency or excess are detrimental for organisms and cells. Therefore, iron and carbohydrate metabolism are tightly regulated. Serum iron and glucose levels are subjected to hormonal regulation by hepcidin and insulin, respectively. Hepcidin is a liver-derived peptide hormone that inactivates the iron exporter ferroportin in target cells, thereby limiting iron efflux to the bloodstream. Insulin is a protein hormone secreted from pancreatic β-cells that stimulates glucose uptake and metabolism via insulin receptor signaling. There is increasing evidence that systemic, but also cellular iron and glucose metabolic pathways are interconnected. This review article presents relevant data derived primarily from mouse models and biochemical studies. In addition, it discusses iron and glucose metabolism in the context of human disease.
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Iron is an essential nutrient that is potentially toxic due to its redox reactivity. Insufficient iron supply to erythroid cells, the major iron consumers in the body, leads to various forms of anemia. On the other hand, iron overload... more
Iron is an essential nutrient that is potentially toxic due to its redox reactivity. Insufficient iron supply to erythroid cells, the major iron consumers in the body, leads to various forms of anemia. On the other hand, iron overload (hemochromatosis) is associated with tissue damage and diseases of liver, pancreas, and heart. Physiological iron balance is tightly controlled at the cellular and systemic level by iron regulatory proteins (IRP1, IRP2) and the iron regulatory hormone hepcidin, respectively. Underlying mechanisms often intersect to achieve optimal iron utilization, to control immune responses, and to prevent iron toxicity. This review focuses on systemic iron homeostasis in the context of erythropoiesis, a highly iron-demanding process. We discuss the function and regulation of hepcidin by various stimuli, and highlight hepcidin-dependent and -independent mechanisms that link iron utilization with maturation of erythroid progenitor cells. © 2017 IUBMB Life, 2017.
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The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. These cytosolic RNA-binding proteins control the synthesis of proteins involved in storage, transport, and... more
The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. These cytosolic RNA-binding proteins control the synthesis of proteins involved in storage, transport, and utilization of iron. Whereas IRP1 levels remain nearly constant, IRP2 is rapidly degraded by the proteasome in iron-replete cells. In non iron-loaded H1299 human lung cancer cells, the decay of transfected hemagglutinin-tagged IRP2 was significantly antagonized by addition of not only proteasomal, but also lysosomal inhibitors. Similar results were obtained with IRP2-Ins5 , a molecular form lacking the specific IRP2 domain of 73 amino acids that is absent from IRP1. These data uncover an alternative, iron independent, mechanism of IRP2 degradation via the lysosomal pathway. Transfected IRP1 decayed slowly over several days and, in contrast to IRP2, was not further stabilized by proteasomal or lysosomal inhibitors. Experiments with an IRP1/IRP2 hybrid molecule and with IRP2 variants indicated that proteins lacking the C-terminus of IRP2 were insensitive to lysosomal inhibitors. Together with previous data obtained in the presence of iron excess, these results show that the parallel degradation pathways through lysosomes and the proteasome that are active on IRP2 under normal growth conditions are preferentially shifted to the proteasome when iron becomes plentiful.
Mutations in the HFE or HJV genes are associated with adult or juvenile forms of hereditary hemochromatosis, respectively, a disease of systemic iron overload that is triggered by defective expression of the iron regulatory hormone... more
Mutations in the HFE or HJV genes are associated with adult or juvenile forms of hereditary hemochromatosis, respectively, a disease of systemic iron overload that is triggered by defective expression of the iron regulatory hormone hepcidin. Two recent publications examined the roles of murine Hfe and Hjv on iron signaling to hepcidin (1, 2). They address whether these genes exhibit overlapping or distinct functions on the hepcidin pathway by generating and characterizing double Hfe-/-Hjv-/- mice. The conclusions reached are seemingly contradictory, even though the experimental data are largely consistent. Both studies report that compound inactivation of Hfe and Hjv does not further aggravate iron overload and hepcidin insufficiency caused by single Hjv ablation. This article is protected by copyright. All rights reserved.
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Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression of proteins implicated in iron and energy metabolism. IRE/IRP1 interactions modulate mRNA translation or stability and result in homeostatic adaptations to... more
Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression of proteins implicated in iron and energy metabolism. IRE/IRP1 interactions modulate mRNA translation or stability and result in homeostatic adaptations to changes in iron availability. IRP1 belongs to the family of iron–sulfur isomerases. Its genetic activity is regulated by the iron dependent assembly–disassembly of a cubane, aconitase-type [4Fe–4S] cluster. Direct administration of hydrogen peroxide to cells leads to a rapid activation of IRP1 to its IRE-binding form—IRPI activation. This chapter describes the basic methods that have been developed and applied to study the activation of IRPI by hydrogen peroxide. These include the electrophoretic mobility shift assay to detect IRE-binding activity, the chemiluminescence luminol/hypochlorite assay to detect extracellular hydrogen peroxide, the method for enzymatic generation of hydrogen peroxide at steady-state levels, and the fluorometric assay to monitor relative intracellular hydrogen peroxide levels. In addition, the chapter describes key experiments that have provided insights regarding the mechanism and the physiological implications of IRP1 activation by hydrogen peroxide in cultured B6 fibroblasts, in permeabilized B6 fibroblasts, and in the intact rat liver.