Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Role of hepcidin in murine brain iron metabolism.

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism.

Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element.

This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)-like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex from P falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

Systemic iron metabolism: a review and implications for brain iron metabolism.

Mammalian cells and organisms coordinate to regulate expression of numerous proteins involved in the uptake, sequestration, and export of iron. When cells in the systemic circulation are depleted of iron, they increase synthesis of the transferrin receptor and decrease synthesis of the iron sequestration protein, ferritin. In iron-depleted animals, expression of duodenal iron transporters markedly increases and intestinal iron uptake increases accordingly. The major proteins of iron metabolism in the systemic circulation are also expressed in the central nervous system. However, the mechanisms by which iron is transported and distributed throughout the central nervous system are not well understood. Iron accumulation in specific regions of the brain is observed in several neurodegenerative diseases. It is likely that misregulation of iron metabolism is important in the pathophysiology of several human neurodegenerative diseases.

Association of pulmonary tuberculosis with increased dietary iron.

To determine whether increased dietary iron could be a risk factor for active tuberculosis, dietary iron history and human immunodeficiency virus (HIV) status were studied in 98 patients with pulmonary tuberculosis and in 98 control subjects from rural Zimbabwe. Exposure to high levels of dietary iron in the form of traditional beer is associated with increased iron stores in rural Africans. HIV seropositivity was associated with a 17.3-fold increase in the estimated odds of developing active tuberculosis (95% confidence interval [95% CI], 7.4-40.6; P<.001), and increased dietary iron was associated with a 3.5-fold increase (95% CI, 1.4-8.9; P=.009). Among patients treated for tuberculosis, HIV seropositivity was associated with a 3.8-fold increase in the estimated hazard ratio of death (95% CI, 1.0-13.8; P=.046), and increased dietary iron was associated with a 1.3-fold increase (95% CI, 0.4-6.4; P=.2). These findings are consistent with the hypothesis that elevated dietary iron may increase the risk of active pulmonary tuberculosis.

Iron on the brain.

Accumulations of iron are often detected in the brains of people suffering from neurodegenerative diseases. But it is often not known whether such accumulations contribute directly to disease progression. The identification of the genes mutated in two such disorders suggests that errors in iron metabolism do indeed have a key role.

MCV as a guide to phlebotomy therapy for hemochromatosis.

BACKGROUND: A multitude of recommendations exist for laboratory assays to monitor the pace and endpoints of phlebotomy therapy for hemochromatosis. All of these recommendations rely on an assessment of storage iron to guide treatment, and none have been prospectively evaluated. STUDY DESIGN AND METHODS: Nine consecutive patients underwent serial monitoring of Hb, MCV, transferrin saturation, and ferritin during weekly phlebotomy to deplete iron stores (induction therapy) and less frequent sessions to prevent iron reaccumulation (maintenance therapy). Changes in MCV and Hb were used to guide the pace of phlebotomy over a median of 7 years of follow-up. RESULTS: During induction therapy, the MCV increased transiently because of reticulocytosis and then stabilized for a prolonged period before decreasing more sharply, which reflected iron-limited erythropoiesis. Iron depletion was achieved after a median of 38 phlebotomies and removal of 9.0 g of iron. Maintenance phlebotomy was targeted to maintain the MCV at 5 to 10 percent below prephlebotomy values and the Hb at >13 g per dL. Transferrin saturation fluctuated considerably during treatment, but remained below 35 percent during MCV-guided maintenance therapy. Ferritin values were not useful guides to the pace of phlebotomy. The median maintenance therapy phlebotomy interval was 7.5 weeks (range, 6-16), which corresponded to an average daily iron removal of 35 to 67 microg per kg. Most patients showed evidence of iron reaccumulation at phlebotomy intervals of 8 weeks or more. CONCLUSION: The MCV is an inexpensive, precise, physiologic indicator of erythropoietic iron availability. When used in conjunction with the Hb, it is a clinically useful guide to the pace of phlebotomy therapy for hemochromatosis.

Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells.

Iron-sulfur (Fe-S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe-S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe-S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe-S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe-S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe-S cluster assembly are discussed.

Regulation of intracellular iron metabolism in human erythroid precursors by internalized extracellular ferritin.

Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with (59)Fe- and/or (125)I-labeled acid isoferritin and chased. The (125)I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, (59)Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.

Measurements of iron status and survival in African iron overload.

INTRODUCTION: Dietary iron overload is common in southern Africa and there is a misconception that the condition is benign. Early descriptions of the condition relied on autopsy studies, and the use of indirect measurements of iron status to diagnose this form of iron overload has not been clarified. METHODS: The study involved 22 black subjects found to have iron overload on liver biopsy. Fourteen subjects presented to hospital with liver disease and were found to have iron overload on percutaneous liver biopsy. Eight subjects, drawn from a family study, underwent liver biopsy because of elevated serum ferritin concentrations suggestive of iron overload. Indirect measurements of iron status (transferrin saturation, serum ferritin) were performed on all subjects. Histological iron grade and hepatic iron concentration were used as direct measures of iron status. RESULTS: There were no significant differences in either direct or indirect measurements of iron status between the two groups. In 75% of these subjects the hepatic iron concentration was greater than 350 micrograms/g dry weight, an extreme elevation associated with a high risk of fibrosis and cirrhosis. Serum ferritin was elevated in all subjects and the transferrin saturation was greater than 60% in 93% of the subjects. Hepatomegaly was present in 20 of the 22 cases and there was only a moderate derangement in liver enzymes except for a tenfold increase in the median gamma-glutamyl transpeptidase concentration. There was a strong correlation between serum ferritin and hepatic iron concentrations (r = 0.71, P = 0.006). After a median follow-up of 19 months, 6 (26%) of the subjects had died. The risk of mortality correlated significantly with both the hepatic iron concentration and the serum ferritin concentration. CONCLUSIONS: Indirect measurements of iron status (serum ferritin concentration and transferrin saturation) are useful in the diagnosis of African dietary iron overload. When dietary iron overload becomes symptomatic it has a high mortality. Measures to prevent and treat this condition are needed.

A traditional beverage prevents iron deficiency in African women of child bearing age.

OBJECTIVE: To determine if a traditional item in the diet might be useful in preventing iron deficiency in African women of child-bearing age. DESIGN: In a prospective study, the iron status of women who did and did not drink traditional beer high in iron and folic acid, was compared. Iron status was determined by a combination of haemoglobin, serum ferritin and transferrin saturation. SETTING: The study was conducted amongst rural villagers in the Murehwa and Zaka districts of Zimbabwe and in Mpumalanga Province, South Africa. SUBJECTS: 112 women aged between 12 and 50 y from a population of 425 rural people participating in on-going family genetic studies. RESULTS: Women who consumed traditional beer had significantly higher serum ferritin concentrations and transferrin saturations compared to non-drinkers (P = 0.0001 and 0.03 respectively). Iron deficiency anaemia was not present in drinkers but the prevalence in non-drinkers was 13%. Forty seven percent of the non-drinkers and only 14% of the drinkers had evidence of iron deficiency (P = 0.002). Six (21%) of the drinkers and none of the non-drinkers had evidence of iron overload (transferrin saturation > 55% and serum ferritin > 400 ug/l). CONCLUSION: We conclude that the consumption of traditional beer, rich in iron, protects women against iron deficiency. While the use of an alcoholic beverage is not ideal, our findings suggest that indigenous cultural practices might be successfully employed or adapted for promoting iron nutrition.

Clinical severity and thermodynamic effects of iron-responsive element mutations in hereditary hyperferritinemia-cataract syndrome.

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a novel genetic disorder characterized by elevated serum ferritin and early onset cataract formation. The excessive ferritin production in HHCS patients arises from aberrant regulation of L-ferritin translation caused by mutations within the iron-responsive element (IRE) of the L-ferritin transcript. IREs serve as binding sites for iron regulatory proteins (IRPs), iron-sensing proteins that regulate ferritin translation. Previous observations suggested that each unique HHCS mutation conferred a characteristic degree of hyperferritinemia and cataract severity in affected individuals. Here we have measured the in vitro affinity of the IRPs for the mutant IREs and correlated decreases in binding affinity with clinical severity. Thermodynamic analysis of these IREs has also revealed that although some HHCS mutations lead to changes in the stability and secondary structure of the IRE, others appear to disrupt IRP-IRE recognition with minimal effect on IRE stability. HHCS is a noteworthy example of a human genetic disorder that arises from mutations within a protein-binding site of an mRNA cis-acting element. Analysis of the effects of these mutations on the energetics of the RNA-protein interaction explains the phenotypic variabilities of the disease state.

Iron overload in urban Africans in the 1990s.

BACKGROUND: In a previously described model, heterozygotes for an African iron loading locus develop iron overload only when dietary iron is high, but homozygotes may do so with normal dietary iron. If an iron loading gene is common, then homozygotes with iron overload will be found even in an urban population where traditional beer, the source of iron, is uncommon. AIMS: To determine whether iron overload and the C282Y mutation characteristic of hereditary haemochromatosis are readily identifiable in an urban African population. METHODS: Histological assessment, hepatocellular iron grading, and dry weight non-haem iron concentration were determined in post mortem tissue from liver, spleen, heart, lungs, and skin. DNA of subjects with elevated hepatic iron indexes was analysed for the C282Y mutation. Iron concentrations in other tissues were compared. RESULTS: A moderate increase (>30 micromol/g) in hepatic iron concentrations was found in 31 subjects (23%; 95% confidence interval 15.9 to 30.1%), and they were considerably elevated (>180 micromol/g) in seven subjects (5.2%; 95% confidence interval 1.5 to 8.9%). Appreciably elevated hepatic iron concentrations were associated with heavy iron deposition in both hepatocytes and macrophages, and either portal fibrosis or cirrhosis. All were negative for the C282Y mutation. Very high concentrations were uncommon in subjects dying in hospital. Concentrations of iron in spleen, heart, lung, and skin were significantly higher in subjects with elevated hepatic iron. CONCLUSIONS: Iron overload is readily identified among urban Africans and is associated with hepatic damage and iron loading of several tissues. The condition is unrelated to the genetic mutation found in hereditary haemochromatosis.

Non-transferrin-bound iron and hepatic dysfunction in African dietary iron overload.

BACKGROUND: Circulating iron is normally bound to transferrin. Non-transferrin-bound iron (NTBI) has been described in most forms of iron overload, but has not been studied in African dietary iron overload. This abnormal iron fraction is probably toxic, but this has not been demonstrated. METHODS: High-pressure liquid chromatography was used to assay serum NTBI in 25 black African subjects with iron overload documented by liver biopsy and in 170 relatives and neighbours. Levels of NTBI were correlated with indirect measures of iron status and conventional liver function tests. RESULTS: Non-transferrin-bound iron (> 2 micromol/L) was present in 43 people, 22 of patients of whom underwent liver biopsy and 21 relatives and neighbours. All but four of these had evidence of iron overload on the basis of either liver biopsy or elevated transferrin and serum ferritin concentrations. Among all 195 subjects, the presence of NTBI in serum was independently related to elevations in alanine and aspartate aminotransferase activity and bilirubin concentration. This relationship between serum NTBI and hepatic dysfunction was confirmed in the subgroup of 25 subjects with iron overload documented by liver biopsy. Non-transferrin-bound iron correlated significantly with elevations in alanine and aspartate aminotransferase activities after adjustment for hepatic iron grades, inflammation and diet. CONCLUSIONS: Non-transferrin-bound iron was found to be commonly present in African patients with dietary iron overload and to correlate with transferrin saturation and serum ferritin concentration. The independent relationship between NTBI and elevated liver function tests suggests that it may be part of a pathway leading to hepatic injury.

Iron and alcohol content of traditional beers in rural Zimbabwe.

OBJECTIVES: To determine the concentrations of iron and alcohol in traditional beer, as well as how these may be related to the brewing process. DESIGN: Cross sectional study. SETTING/SUBJECTS: Rural communities living in four of Zimbabwe's nine provinces. MAIN OUTCOME MEASURES: Ionic iron concentration and alcohol concentration in 94 different types of alcoholic beverages prepared in rural areas, and 18 commercially produced beers. RESULTS: The commonest types of traditional beer were a seven day beverage called 'doro rematanda', a by-product of this seven day beer called 'muchaiwa,' and a one-day beverage called 'chikokiyana'. Methods of preparation were similar in the four provinces. Median (Q1, Q3) ionic iron concentrations were 52 (31 to 75) mg/L for the seven-day beer (n = 51), 24 (18 to 36) mg/L for muchaiwa (n = 30) and 21 (17 to 63) mg/L for chikokiyana (n = 13). In contrast, ionic iron concentrations in 12 samples of commercially prepared clear beers were 0.1 mg/L and in commercial opaque beer were 3.6 mg/L. Mean (SD) alcohol concentration in traditional beer was 4.1 g/100 ml (+/- 0.873) compared to 2.8 g/100 ml +/- 1.394) in the muchaiwa and 3.6 g/100 ml (+/- 1.445) in the one day brew, chikokiyana. Mean alcohol concentrations in the three commercial beers are reportedly 3.5 g/100 ml in the opaque beer (Scud), and 4.7 to 5.0 g/ml in clear beer (Zambezi and Castle lagers). CONCLUSIONS: Several preparation methods lead to traditional fermented beverages with very high iron concentrations. Measures to prevent dietary iron overload should include all of these beverages in their scope.

Is there a link between African iron overload and the described mutations of the hereditary haemochromatosis gene?

Over 80%, of Caucasian patients with hereditary haemochromatosis are homozygotes for a C282Y mutation in the HFE gene on chromosome 6. Recent evidence suggests that a genetic factor may also be involved in the pathogenesis of African iron overload, although the locus has not been described. PCR analysis of DNA from 25 southern Africans, identified by segregation analysis as having a high probability of carrying the putative African iron-loading gene, failed to identify any subjects with the C282Y mutation. The possible genetic defect in African iron overload appears to be different from that described in most cases of hereditary haemochromatosis in Caucasians.

Cell-cycle arrest and inhibition of G1 cyclin translation by iron in AFT1-1(up) yeast.

Although iron is an essential nutrient, it is also a potent cellular toxin, and the acquisition of iron is a highly regulated process in eukaryotes. In yeast, iron uptake is homeostatically regulated by the transcription factor encoded by AFT1. Expression of AFT1-1(up), a dominant mutant allele, results in inappropriately high rates of iron uptake, and AFT1-1(up) mutants grow slowly in the presence of high concentrations of iron. We present evidence that when Aft1-1(up) mutants are exposed to iron, they arrest the cell division cycle at the G1 regulatory point Start. This arrest is dependent on high-affinity iron uptake and does not require the activation of the DNA damage checkpoint governed by RAD9. The iron-induced arrest is bypassed by overexpression of a mutant G1 cyclin, cln3-2, and expression of the G1-specific cyclins Cln1 and Cln2 is reduced when yeast are exposed to increasing amounts of iron, which may account for the arrest. This reduction is not due to changes in transcription of CLN1 or CLN2, nor is it due to accelerated degradation of the protein. Instead, this reduction occurs at the level of Cln2 translation, a recently recognized locus of cell-cycle control in yeast.