Iron-storage Ferritin Research Articles

Amyotrophic lateral sclerosis associated disturbance of iron metabolism is blunted by swim training-role of AKT signaling pathway

Swim training has increased the life span of the transgenic animal model of amyotrophic lateral sclerosis (ALS). Conversely, the progress of the disease is associated with the impairment of iron metabolism and insulin signaling.We used transgenic hmSOD1 G93A (ALS model) and non-transgenic mice in the present study. The study was performed on the muscles taken from trained (ONSET and TERMINAL) and untrained animals at three stages of the disease: BEFORE, ONSET, and TERMINAL. In order to study the molecular mechanism of changes in iron metabolism, we used SH-SY5Y and C2C12 cell lines expression vector pcDNA3.1 and transiently transfected with specific siRNAs.The progress of ALS resulted in decreased P-Akt/Akt ratio, which is associated with increased proteins responsible for iron storage ferritin L, ferritin H, PCBP1, and skeletal muscle iron at ONSET. Conversely, proteins responsible for iron export- TAU significantly decrease. The training partially reverses changes in proteins responsible for iron metabolism. AKT silencing in the SH-SY5Y cell line decreased PCBP2 and ferroportin and increased ferritin L, H, PCBP1, TAU, transferrin receptor 1, and APP. Moreover, silencing APP led to an increase in ferritin L and H.Our data suggest that swim training in the mice ALS model is associated with significant changes in iron metabolism related to AKT activity. Down-regulation of AKT mainly upregulates proteins involved in iron import and storage but decreases proteins involved in iron export.

The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-kowt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-koko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-koko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-kowt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-koko-BM=174.8±50.4 μg/g; Ncoa4-kowt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-koko-BM=719.0±221.3 μg/g; Ncoa4-kowt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-koko-BM animals.Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Room-temperature tunnel magnetoresistance across biomolecular tunnel junctions based on ferritin

We report exceptionally large tunnel magnetoresistance (TMR) for biomolecular tunnel junctions based on ferritins immobilized between Ni and EGaIn electrodes. Ferritin stores iron in the form of ferrihydrite nanoparticles (NPs) and fulfills the following roles: (a) it dictates the tunnel barrier, (b) it magnetically decouples the NPs from the ferromagnetic (FM) electrode, (c) it stabilizes the NPs, and (d) it acts as a spin filter reducing the complexity of the tunnel junctions since only one FM electrode is required. The mechanism of charge transport is long-range tunneling which results in TMR of 60 ± 10% at 200 K and 25 ± 5% at room temperature. We propose a magnon-assisted transmission to explain the substantially larger TMR switching fields (up to 1 Tesla) than the characteristic coercive fields (a few Gauss) of ferritin ferrihydrite particles at T < 20 K. These results highlight the genuine potential of biomolecular tunnel junctions in designing functional nanoscale spintronic devices.

Aerosolized Bovine Lactoferrin Counteracts Infection, Inflammation and Iron Dysbalance in A Cystic Fibrosis Mouse Model of Pseudomonas aeruginosa Chronic Lung Infection.

Cystic fibrosis (CF) is a genetic disorder affecting several organs including airways. Bacterial infection, inflammation and iron dysbalance play a major role in the chronicity and severity of the lung pathology. The aim of this study was to investigate the effect of lactoferrin (Lf), a multifunctional iron-chelating glycoprotein of innate immunity, in a CF murine model of Pseudomonas aeruginosa chronic lung infection. To induce chronic lung infection, C57BL/6 mice, either cystic fibrosis transmembrane conductance regulator (CFTR)-deficient (Cftrtm1UNCTgN(FABPCFTR)#Jaw) or wild-type (WT), were intra-tracheally inoculated with multidrug-resistant MDR-RP73 P. aeruginosa embedded in agar beads. Treatments with aerosolized bovine Lf (bLf) or saline were started five minutes after infection and repeated daily for six days. Our results demonstrated that aerosolized bLf was effective in significantly reducing both pulmonary bacterial load and infiltrated leukocytes in infected CF mice. Furthermore, for the first time, we showed that bLf reduced pulmonary iron overload, in both WT and CF mice. In particular, at molecular level, a significant decrease of both the iron exporter ferroportin and iron storage ferritin, as well as luminal iron content was observed. Overall, bLf acts as a potent multi-targeting agent able to break the vicious cycle induced by P. aeruginosa, inflammation and iron dysbalance, thus mitigating the severity of CF-related pathology and sequelae.

Impact of Inflammation on Ferritin, Hepcidin and the Management of Iron Deficiency Anemia in Chronic Kidney Disease.

Iron deficiency anemia (IDA) is a major problem in chronic kidney disease (CKD), causing increased mortality. Ferritin stores iron, representing iron status. Hepcidin binds to ferroportin, thereby inhibiting iron absorption/efflux. Inflammation in CKD increases ferritin and hepcidin independent of iron status, which reduce iron availability. While intravenous iron therapy (IIT) is superior to oral iron therapy (OIT) in CKD patients with inflammation, OIT is as effective as IIT in those without. Inflammation reduces predictive values of ferritin and hepcidin for iron status and responsiveness to iron therapy. Upper limit of ferritin to predict iron overload is higher in CKD patients with inflammation than in those without. However, magnetic resonance imaging studies show lower cutoff levels of serum ferritin to predict iron overload in dialysis patients with apparent inflammation than upper limit of ferritin proposed by international guidelines. Compared to CKD patients with inflammation, optimal ferritin levels for IDA are lower in those without, requiring reduced iron dose and leading to decreased mortality. The management of IDA should differ between CKD patients with and without inflammation and include minimization of inflammation. Further studies are needed to determine the impact of inflammation on ferritin, hepcidin and therapeutic strategy for IDA in CKD.

Intracellular Biodegradation of Ag Nanoparticles, Storage in Ferritin, and Protection by a Au Shell for Enhanced Photothermal Therapy.

Despite their highly efficient plasmonic properties, gold nanoparticles are currently preferred to silver nanoparticles for biomedical applications such as photothermal therapy due to their high chemical stability in the biological environment. To confer protection while preserving their plasmonic properties, we allied the advantages of both materials and produced hybrid nanoparticles made of an anisotropic silver nanoplate core coated with a frame of gold. The efficiency of these hybrid nanoparticles (Ag@AuNPs) in photothermia was compared to monometallic silver nanoplates (AgNPs) or gold nanostars (AuNPs). The structural and functional properties of AuNPs, AgNPs, and Ag@AuNPs were investigated in environments of increasing complexity, in water suspensions, in cells, and in tumors in vivo. While AgNPs showed the greatest heating efficiency in suspension (followed by Ag@AuNPs and AuNPs), this trend was reversed intracellularly within a tissue-mimetic model. In this setup, AgNPs failed to provide consistent photothermal conversion over time, due to structural damage induced by the intracellular environment. Remarkably, the degraded Ag was found to be stored within the iron-storage ferritin protein. By contrast, the Au shell provided the Ag@AuNPs with total Ag biopersistence. As a result, photothermal therapy was successful with Ag@AuNPs in vivo in a mouse tumor model, providing the ultimate proof on Au shell's capability to shield the Ag core from the harsh biological environment and preserve its excellent heating properties.

Ostreococcus tauri Luminescent Reporter Lines as Biosensors for Detecting Pollution From Copper-Mine Tailing Effluents in Coastal Environments

Phytoplankton cells are excellent biosensors for environmental monitoring and toxicity assessments in different natural systems. Green algae, in particular, appear to be more responsive to copper (Cu) disturbances. This is interesting considering that Cu pollution in coastal environments has increased over the last century, with enormous repercussions to marine ecosystems. Unfortunately, no high-throughput method exists for the environmental monitoring of Cu toxicity in seawater. To assess potential uses as biosensors of Cu pollution, high-throughput screening was performed on five luminescence reporter lines constructed in the green algae Ostreococcus tauri RCC745. The reporter line expressing the iron storage ferritin protein fused to luciferase (Fer-Luc) was the most sensitive, responding to Cu concentrations in the µM range. Fer-Luc was also the most sensitive reporter line for detecting toxicity in mining-derived polluted seawater predominantly contaminated by soluble Cu. Nevertheless, the Cyclin-Dependent-Kinase A (CDKA) reporter was most suitable for detecting the toxicity of copper-mine tailing effluents containing other metals (e.g., iron). These results highlight that Ostreococcus biosensors can serve as a reliable, inexpensive, and automated, high-throughput laboratory approach for performing seawater analyses of coastal areas subjected to metal disturbances. When challenged with Cu, Ostreococcus tauri not only evidenced a rapid, transcriptional response for the tested genes, but also showed changes in a broad range of genes, especially as related to the stress response. Overall, the obtained results reinforce that a single biosensor is insufficient when dealing with complex mixtures of toxic compounds in natural environments.

P-18 - Iron-homeostasis regulation by neuronal oxygen sensors in the nematode Caenorhabditis elegans

Iron presents cells with a double-edged sword. On the one hand, iron is crucial for fundamental physiological processes such as DNA synthesis, oxygen (O2) transport, and mitochondrial respiration. On the other hand, iron can catalyze the formation of toxic oxygen radicals that can destroy any biological molecule. Therefore, it is crucial to understand how the concentration of labile iron is regulated at the whole animal level. And, in particular, how it is controlled in stress conditions in which the level of reactive oxygen species (ROS) is high. Here, we characterized the changes in intestinal gene expression occurring during the adaptation of C. elegans worms to hypoxia and reoxygenation stress. Among the 770 genes that were differentially expressed, the iron storage ferritin 1 (ftn-1) gene was significantly upregulated in hypoxia. Intriguingly, we show that this upregulation is controlled by neuronal oxygen-sensors in a HIF-1-dependent manner. Moreover, our data suggest that FTN-1 plays an important role in innate immunity in hypoxia. These data may provide novel insights about the way iron level is regulated in tumor development where cells may experience mild to severe hypoxia conditions.

Engineering intracellular biomineralization and biosensing by a magnetic protein.

Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 107 variants induces threefold greater cellular iron loading than mammalian heavy chain Ft, over fivefold higher contrast in magnetic resonance imaging, and robust retention on magnetic separation columns. Mechanistic studies of mutant Ft proteins indicate that improved magnetism arises in part from increased iron oxide nucleation efficiency. Molecular-level iron loading in engineered Ft enables detection of individual particles inside cells and facilitates creation of Ft-based intracellular magnetic devices. We demonstrate construction of a magnetic sensor actuated by gene expression in yeast.

The crystal structure of ferritin from Chlorobium tepidum reveals a new conformation of the 4-fold channel for this protein family.

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3Å; corresponding to a diferric μ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.

Transferrin Deficiency Leads To Specific and Partially Reversible Iron Overload

Studies in hpx mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery to the erythron and regulation of expression of hepcidin, a hormone that inhibits dietary iron absorption. Here we address two other putative roles for s transferrin. First, we determined if transferrin is essential for metabolism of metals other than iron. Metal content analysis of fractionated mouse sera indicated that iron was the most abundant metal in transferrin-rich fractions. Organ metal content analysis demonstrated severe imbalances for iron and minimal to moderate imbalances for copper, zinc and manganese in hpx tissues. Analysis of metal-dependent gene expression and organ metal content in a mouse model of inherited hepcidin excess suggested that hpx tissue imbalances in copper, zinc and manganese levels were not all physiologic or specific to primary transferrin deficiency. Second, we tested if transferrin modulates the mobilization of tissue iron stores. Transferrin treatment of hpx mice decreased iron levels in most tissues analyzed. Iron mobilization was independent of hepcidin, as iron levels decreased in hpx mice deficient in hemojuvelin, a membrane protein essential for hepcidin expression. Iron mobilization led to decreased ferritin iron and heavy chain levels. The decrease in liver ferritin iron stores did not match the decrease in total liver iron stores, suggesting that iron was mobilized from multiple storage sites. Overall, our analysis suggests that the primary consequences of transferrin deficiency are largely specific to iron and that transferrin can modulate the mobilization of tissue iron stores. Disclosures:No relevant conflicts of interest to declare.

Identification of an Arabidopsis mitoferrinlike carrier protein involved in Fe metabolism

Iron has a major role in mitochondrial as well as in chloroplast metabolism, however the processes involved in organelle iron transport in plants are only partially understood. To identify mitochondrial iron transporters in Arabidopsis, we searched for proteins homologous to the Danio rerio (zebrafish) Mitoferrin2 MFRN2, a mitochondrial iron importer in non-erythroid cells. Among the identified putative Arabidopsis mitoferrinlike proteins, we focused on that one encoded by At5g42130, which we named AtMfl1 (MitoFerrinLike1). AtMfl1 expression strongly correlates with genes coding for proteins involved in chloroplast metabolism. Such an unexpected result is supported by the identification by different research groups, of the protein encoded by At5g42130 and of its homologs from various plant species in the inner chloroplastic envelope membrane proteome. Notably, neither the protein encoded by At5g42130 nor its homologs from other plant species have been identified in the mitochondrial proteome. AtMfl1 gene expression is dependent on Fe supply: AtMfl1 transcript strongly accumulates under Fe excess, moderately under Fe sufficiency and weakly under Fe deficiency. In order to understand the physiological role of AtMfl1, we isolated and characterized two independent AtMfl1 KO mutants, atmfl1-1 and atmfl1-2: both show reduced vegetative growth. When grown under conditions of Fe excess, atmfl1-1 and atmfl1-2 mutants (seedlings, rosette leaves) contain less total Fe than wt and also reduced expression of the iron storage ferritin AtFer1. Taken together, these results suggest that Arabidopsis mitoferrinlike gene AtMfl1 is involved in Fe transport into chloroplasts, under different conditions of Fe supply and that suppression of its expression alters plant Fe accumulation in various developmental stages.

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.

Age-associated increases in heme oxygenase-1 and ferritin immunoreactivity in the autopsied brain

Heme oxygenase-1 (HO-1) is a 32 kDa heat shock protein (HSP) that catalyzes heme to biliverdin, free iron and carbon monoxide in the brain. Furthermore, the release of free ferrous ion by HO-1 plays an essential role in ferritin synthesis, and ferritin stores iron either for intracellular utilization, or for detoxification. It is well known that HO-1 immunoreactivity is enhanced greatly in neurons and glia of the hippocampus and cerebral cortex in various pathophysiological conditions. The expression of HSP 70 is well known for the age-associated increase, but the expression modalities of HO-1 and ferritin associated with aging are still unknown. A study was therefore performed to examine the correlations in the expression of HO-1 and ferritin with age using immunohistochemistry. We investigated 31 autopsied brains (3–84-year-olds) without traumatic brain injury and neurodegenerative disease. The specimens were taken from the cerebral cortex and hippocampus. In the cerebral cortex, age (aging) had a statistically significant positive correlation with HO-1 ( r=0.894, P<0.01) and ferritin ( r=0.731, P<0.01). In the hippocampus, age had a significant positive correlation with only HO-1 ( r=0.660, P<0.01). These results showed that HO-1 and ferritin underwent an age-related increase in human brain, especially in the cerebral cortex. Our results also indicate that various stress responses may persist in the aged human brain.

Sulfide is an efficient iron releasing agent for mammalian ferritins

The most prominent role of mammalian ferritins is to provide an extensive iron-buffering capacity to cells. The large ferritin iron stores can be mobilized in vitro, but the functional relevance of the most efficient iron releasing agents remains elusive. Sulfide is a strongly reducing chemical generated by a series of enzymes. In the presence of limited amounts of sulfide a continuous rate of iron release from ferritin was observed and a majority of the protein iron core was recovered in solution. The rate constants for iron efflux triggered by several reducing or chelating compounds have been measured and compared. Although not as efficient as reduced flavins, sulfide displayed kinetic parameters which suggest a potential physiological role for the chalcogenide in converting the iron storage protein into apoferritin. To further probe the relevance of sulfide in the mobilization of iron, several enzymes, such as NifS, rhodanese, or sulfite reductase generating reduced forms of sulfur by different mechanisms, have been assayed for their ability to catalyze the release of iron from ferritin. The results show that full reduction of sulfur into sulfide is needed to deplete iron from ferritin. These reactions suggest links between sulfur metabolism and intracellular iron homeostasis.

Impaired iron homeostasis in Parkinson's disease.

Despite physiological systems designed to achieve iron homeostasis, increased concentrations of brain iron have been demonstrated in a range of neurodegenerative diseases. These including the parkinsonian syndromes, the trinucleotide repeat disorders and the dementia syndromes. The increased brain iron is confined to those brain regions most affected by the degeneration characteristic of the particular disorder and is suggested to stimulate cell damage via oxidative mechanisms. Changes in central iron homeostasis have been most closely investigated in PD, as this disorder is well characterised both clinically and pathologically. PD is associated with a significant increase in iron in the degenerating substantia nigra (SN) and is measureable in living PD patients and in post-mortem brain. This increase, however, occurs only in the advanced stages of the disease, suggesting that this phenonoma may be a secondary, rather than a primary initiating event, a hypothesis also supported by evidence from animal experiments. The source of the increased iron is unknown but a variety of changes in iron homeostasis have been identified in PD, both in the brain and in the periphery. The possibility that an increased amount of iron may be transported into the SN is supported by data demonstrating that one form of the iron-binding glycoprotein transferrin family, lactotransferrin, is increased in surviving neurons in the SN in the PD brain and that this change is associated with increased numbers of lactotransferrin receptors on neurons and microvessels in the parkinsonian SN. These changes could represent one mechanism by which iron might concentrate within the PD SN. Alternatively, the measured increased in iron might result from a redistribution of ferritin iron stores. Ferritin is located in glial cells while the degenerating neurons do not stain positive for ferritin. As free radicals are highly reactive, it is unlikely that glial-derived free radicals diffuse across the intracellular space in sufficent quantities to damage neuronal constituents. If intracellular iron release contributes to neuronal damage it seems more probable that an intraneuronal iron source is responsible for oxidant-mediated damage. Such a iron source is neuromelanin (NM), a dark-coloured pigment found in the dopaminergic neurons of the human SN. In the normal brain, NM has the ability to bind a variety of metals, including iron, and increased NM-bound iron is reported in the parkinsonian SN. The consequences of these phenomena for the cell have not yet been clarified. In the absence of significant quantities of iron NM can act as an antioxidant, in that it can interact with and inactivate free radicals. On the other hand, in the presence of iron NM appears to act as a proxidant, increasing the rate of free radical production and thus the oxidative load within the vulnerable neurons. Given that increased iron is only apparent in the advanced stages of the disease it is unlikely that NM is of importance for the primary aetiology of PD. A localised increase in tissue iron and its interaction with NM may be, however, important as a secondary mechanism by increasing the oxidative load on the cell, thereby driving neurodegeneration.

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.

How does the ferritin core form?

Ferritin stores iron as ferrihydrite inside a shell composed of H and L protein chains. H chains contain ferroxidase centres catalysing Fe2+ oxidation, while L chains lack these centres but seem to promote ferrihydrite nucleation. Mossbauer spectroscopic studies of recombinant H-chain ferritins show the following: (1) fast Fe2+ oxidation is associated with the formation of Fe3+ dimers at the ferroxidase centre; (2) within 30 min, a portion of the dimers have split to Fe3+ monomers and some monomers have moved to the threefold channels; (3) over longer times, a trend dimer → monomer → core is established. Core formation is accelerated if L chains are present.