Publisher Summary Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression ... 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.
Hepcidin is a liver‐derived peptide hormone that limits iron egress from tissues to the bloodstre... 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.
Despite advances in our knowledge and attempts to improve therapies, β-thalassemia remains a prev... 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...
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT Iron is an essential nutrient but also a potential biohazard. Elaborate homeostatic mech... 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.
Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amoun... 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...
Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron e... 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...
Publisher Summary Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression ... 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.
Hepcidin is a liver‐derived peptide hormone that limits iron egress from tissues to the bloodstre... 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.
Despite advances in our knowledge and attempts to improve therapies, β-thalassemia remains a prev... 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...
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT Iron is an essential nutrient but also a potential biohazard. Elaborate homeostatic mech... 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.
Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amoun... 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...
Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron e... 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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