Iron Regulatory Factor Research Articles

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Comparison between liquid chromatography/tandem mass spectroscopy and a novel latex agglutination method for measurement of hepcidin-25 concentrations in dialysis patients with renal anemia: A multicenter study

Background and aimsHepcidin-25 is an iron regulatory factor that plays an important role in anemia in patients with chronic kidney disease. Although liquid chromatography/tandem mass spectroscopy (LC-MS/MS) is the gold standard for measuring hepcidin-25 concentrations, results cannot be obtained immediately at clinical sites. In contrast, the latex immunoassay (LIA) can be performed using general clinical laboratory equipment, and the results can be obtained quickly. The aim of this study was to evaluate hepcidin-25 concentrations obtained by LC-MS/MS and a novel LIA method and compare the two methods. Materials and methodsHepcidin-25 was measured by LIA and LC-MS/MS in 182 hemodialysis patients. A hepcidin-25-specific reagent and an automatic analyzer were used for LIA, and a commercially available system was used for LC-MS/MS. The Passing-Bablok regression analysis method was used. ResultsOn Passing-Bablok regression analysis, the slope was 1.000, with an intercept of 0.359. Very strong associations were obtained, and the measured values were almost identical. ConclusionThe hepcidin-25 concentrations measured using LIA and those measured by LC-MS/MS were significantly correlated. LIA can be performed using general clinical examination equipment and has a higher throughput than LC-MS/MS. Therefore, measurement of hepcidin-25 concentrations by LIA can be useful for routine laboratory testing.

Iron Depletion in Systemic and Muscle Compartments Defines a Specific Phenotype of Severe COPD in Female and Male Patients: Implications in Exercise Tolerance.

We hypothesized that iron content and regulatory factors, which may be involved in exercise tolerance, are differentially expressed in systemic and muscle compartments in iron deficient severe chronic obstructive pulmonary disease (COPD) patients. In the vastus lateralis and blood of severe COPD patients with/without iron depletion, iron content and regulators, exercise capacity, and muscle function were evaluated in 40 severe COPD patients: non-iron deficiency (NID) and iron deficiency (ID) (20 patients/group). In ID compared to NID patients, exercise capacity, muscle iron and ferritin content, serum transferrin saturation, hepcidin-25, and hemojuvelin decreased, while serum transferrin and soluble transferrin receptor and muscle IRP-1 and IRP-2 increased. Among all COPD, a significant positive correlation was detected between FEV1 and serum transferrin saturation. In ID patients, significant positive correlations were detected between serum ferritin, hepcidin, and muscle iron content and exercise tolerance and between muscle IRP-2 and serum ferritin and hepcidin levels. In ID severe COPD patients, iron content and its regulators are differentially expressed. A potential crosstalk between systemic and muscle compartments was observed in the ID patients. Lung function and exercise capacity were associated with several markers of iron metabolism regulation. Iron status should be included in the overall assessment of COPD patients given its implications in their exercise performance.

Regulation of plant iron homeostasis by abscisic acid: a review

Iron (Fe) is an important trace element involved in many important plant physiological and metabolic processes such as photosynthesis, respiration and nitrogen metabolism. Plants maintain iron homeostasis through absorption, transporting, storage and redistribution of iron. Iron metabolism is strictly regulated in plants. Iron regulatory transcription factors and iron transporters constitute the regulatory network of plant iron absorption and transport in plants. Ferritin and iron transporter jointly regulate the response to excess iron in plants. In recent years, important progress has been made in understanding how abscisic acid (ABA) regulates iron metabolism in plants. ABA may be used as a signal to regulate the absorption, transportation and reuse of Fe, or to relieve the symptoms of iron stress by regulating the oxidative stress responses in plants. In order to gain deeper insights into the crosstalk of ABA and iron metabolism in plants, this review summarized the mechanisms of iron absorption and transport and metabolic regulatory network in plants, as well as the mechanisms of ABA in regulating iron metabolism. The relationship between ABA and FER-like iron deficiency-induced transcription factor (FIT), iron-regulated transporter 1 (IRT1), and oxidative stress of iron deficiency were highlighted, and future research directions were prospected.

Iron Dyshomeostasis Participated in Rat Hippocampus Toxicity Caused by Aluminum Chloride.

Aluminum (Al) alters iron regulatory factors content and leads to the changes in iron-related proteins causing iron accumulation. But limited evidence ascertains this hypothesis. Therefore, our experiment was conducted and four groups of male Wistar rats were orally administrated of 0, 50, 150, and 450mg/kg BW/d aluminum chloride (AlCl3) for 90days by drinking water, respectively. The cognitive function, pathological lesion of hippocampus, oxidative stress, as well as iron-related proteins and iron regulatory factors expression were detected. The results showed that AlCl3 remarkably induced the oxidative stress and pathological lesion in the hippocampus and impaired the learning-memory ability. The contents of Al and iron increased in all AlCl3-exposed groups. Meanwhile, the increased divalent metal transporter 1 (DMT1) expression enhanced iron import and the decreased ferroportin 1 (Fpn1) expression reduced iron export in AlCl3-exposed groups. The iron accumulated and ferritin heavy chains (Fth) expression decreased in all AlCl3-exposed groups led to an increase in free iron. The study also showed that iron regulatory factor iron regulatory protein 2 (IRP2) was decreased and hepcidin was increased in AlCl3-exposed groups. The results indicated that AlCl3 induces iron dyshomeostasis presenting as iron accumulation, the disordered expression of iron import, export, store, and regulatory proteins in rat hippocampus accompanied with oxidative stress, pathological lesion, and impaired learning-memory ability.

Six-Transmembrane Epithelial Antigen of Prostate 3 Predicts Poor Prognosis and Promotes Glioblastoma Growth and Invasion

Recent evidence suggests that dysregulation of iron regulatory factors may play essential roles in cancer pathophysiology. Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is a metalloreductase, which is vital for cellular iron uptake and homeostasis. However, the clinical significance and function of STEAP3 in the development of human gliomas remain unclear. Through analysis of publicly available databases, we found that STEAP3 was highly expressed in malignant gliomas, especially in the mesenchymal glioma molecular subtype and isocitrate dehydrogenase 1/2 (IDH1/2) wild-type gliomas. Expression levels of STEAP3 in gliomas correlated inversely with patient overall survival (OS) and served as an independent prognostic marker by multivariate Cox regression analysis. In functional assays performed with RNA knockdown, loss of STEAP3 attenuated aggressive phenotypes in glioma cells, including cell proliferation, invasion, and sphere formation in vitro and tumor growth in vivo. Finally, STEAP3 drives these activities by inducing mesenchymal transition, promoting transferrin receptor (TfR) expression, and activating STAT3-FoxM1 axis signaling. Taken together, these results indicate that STEAP3 functions as an oncogenic mediator in glioma progression and is thus a potential therapeutic target for the treatment of the disease.

Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

ABSTRACTPseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections.

Dysregulation of iron and copper homeostasis in nonalcoholic fatty liver.

Elevated iron stores as indicated by hyperferritinemia with normal or mildly elevated transferrin saturation and mostly mild hepatic iron deposition are a characteristic finding in subjects with non-alcoholic fatty liver disease (NAFLD). Excess iron is observed in approximately one third of NAFLD patients and is commonly referred to as the "dysmetabolic iron overload syndrome". Clinical evidence suggests that elevated body iron stores aggravate the clinical course of NAFLD with regard to liver-related and extrahepatic disease complications which relates to the fact that excess iron catalyses the formation of toxic hydroxyl-radicals subsequently resulting in cellular damage. Iron removal improves insulin sensitivity, delays the onset of type 2 diabetes mellitus, improves pathologic liver function tests and likewise ameliorates NAFLD histology. Several mechanisms contribute to pathologic iron accumulation in NAFLD. These include impaired iron export from hepatocytes and mesenchymal Kupffer cells as a consequence of imbalances in the concentrations of iron regulatory factors, such as hepcidin, cytokines, copper or other dietary factors. This review summarizes the knowledge about iron homeostasis in NAFLD and the rationale for its therapeutic implications.

The Transcriptional Landscape of Campylobacter jejuni under Iron Replete and Iron Limited Growth Conditions

The genome-wide Campylobacter jejuni transcriptional response under iron replete and iron limited conditions was characterized using RNA-seq. We have identified 111 novel C. jejuni 5’UTRs and mapped 377 co-transcribed genes into 230 transcriptional operons. In contrast to previous microarray results, the C. jejuni iron stimulon is less extensive than previously believed and consists of 77 iron activated genes and 50 iron repressed genes. As anticipated, the iron repressed genes are primarily those involved in iron acquisition or oxidative stress defense. Interestingly, these experiments have revealed that iron is an important modulator of flagellar biogenesis with almost all the components of the flagella found to be iron activated. Given that motility is a well-known C. jejuni colonization factor, this suggests that there is an important regulatory coupling of flagellar biogenesis and iron level in C. jejuni. In addition we have identified several consensus mutations in the C. jejuni NCTC11168 strain that are widespread in the Campylobacter research community and which may explain conflicting phenotypic reports for this strain. Comparative analysis of iron responsive genes with the known Fur regulon indicates that many iron responsive genes are not Fur responsive; suggesting that additional iron regulatory factors remain to be characterized in C. jejuni. Further analysis of the RNA-seq data identified multiple novel transcripts including 19 potential ncRNAs. The expression of selected ncRNAs was confirmed and quantified with qRT-PCR. The qRT-PCR results indicate that several of these novel transcripts are either Fur and/or iron responsive. The fact that several of these ncRNAs are iron responsive or Fur regulated suggests that they may perform regulatory roles in iron homeostasis.

Superinfection in malaria: Plasmodium shows its iron will

After the bite of a malaria-infected mosquito, the Plasmodium sporozoite infects liver cells and produces thousands of merozoites, which then infect red blood cells, causing malaria. In malaria-endemic areas, several hundred infected mosquitoes can bite an individual each year, increasing the risk of superinfection. However, in infants that are yet to acquire immunity, superinfections are infrequent. We have recently shown that blood-stage parasitaemia, above a minimum threshold, impairs the growth of a subsequent sporozoite infection of liver cells. Blood-stage parasites stimulate the production of the host iron-regulatory factor hepcidin, which redistributes iron away from hepatocytes, reducing the development of the iron-dependent liver stage. This could explain why Plasmodium superinfection is not often found in young nonimmune children. Here, we discuss the impact that such protection from superinfection might have in epidemiological settings or in programmes for controlling malaria, as well as how the induction of hepcidin and redistribution of iron might influence anaemia and the outcome of non-Plasmodium co-infections.

HapX Positively and Negatively Regulates the Transcriptional Response to Iron Deprivation in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

Psychological stress induces dysregulation of iron metabolism in rat brain

Oxidative damage induced by abnormal iron accumulation in the brain is a primary cause of many neurodegenerative diseases, while the reason for iron deposition remains unclear. A previous study reported that various kinds of stress could cause a change in iron level and psychological stress (PS) was a risk factor for neuron death. In the present study we investigated the influence of PS on iron metabolism in rat brain. The results showed that both total iron and non-protein-bound-iron (NPBI) levels were higher in the cerebral cortex, hippocampus and striatum of PS rats. The levels of iron regulatory factors, including transferrin receptor 1 (TfR1), ferritin (Fn), and iron regulatory protein1 (IRP1), were all changed in the iron deposition regions of the PS-exposed rat brain, accompanied by intensified oxidative stress. It is concluded that PS can increase the intake of iron in some regions of brain and subsequently causes regional iron accumulation, indicating PS might be an important reason for iron deposition–caused neurodegenerative diseases.

Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin

Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.

Regulation of Saccharomyces cerevisiae FET4 by Oxygen and Iron

Saccharomyces cerevisiae expresses two distinct iron transport systems under aerobic and anaerobic conditions. The high affinity transporters, Ftr1p and Fet3p, are primarily expressed in oxygenated cultures, whereas anaerobic conditions induce the low affinity iron transporter, Fet4p. The oxygen regulation of FET4 was found to involve the Rox1p transcriptional repressor. The physiological significance of this control by Rox1p is twofold. First, FET4 repression by Rox1p under oxygenated conditions helps minimize metal toxicity. Sensitivity towards cadmium was high in either anaerobically grown wild-type yeast or in oxygenated rox1Δ strains, and in both cases cadmium toxicity was reversed by FET4 mutations. Secondly, the loss of Rox1p repression under anaerobic conditions serves to induce FET4 and facilitate continual accumulation of iron. We noted that fet4 mutants accumulate lower levels of iron under anaerobic conditions. Regulation of FET4 was examined using FET4- lacZ reporters. We found that FET4 contains a complex promoter regulated both by oxygen and iron status. The region surrounding ≈−960 to −490 contains two consensus Rox1p binding sites and mediates Rox1p, but not iron control of FET4. Sequences downstream of −490 harbor a consensus binding site for the iron regulatory factor Aft1p that is essential for iron regulation in wild-type strains. In addition, a secondary mode of iron regulation becomes evident in strains lacking AFT1. The induction by iron limitation in conjunction with low oxygen is more than additive, suggesting that these activities are synergistic. Fet4p is not the only metal transporter that is negatively regulated by oxygen; we find that Rox1p also represses S. cerevisiae SMF3 , proposed to function in vacuolar iron transport. This oxygen control of iron transporter gene expression is part of an adaptation response to changes in the redox state of transition metals.

The distinct methods by which manganese and iron regulate the Nramp transporters in yeast

The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3—lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt −435 to −350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt −348 to −247 that do not contain obvious Aft1 binding sites. The −348 to −247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.

The distinct methods by which manganese and iron regulate the Nramp transporters in yeast.

The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3-lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt -435 to -350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt -348 to -247 that do not contain obvious Aft1 binding sites. The -348 to -247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.

Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S](+)-containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but not identical to that of m-aconitase. Whilst the [Fe-4S] clusters from both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconitases. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]+ cluster which is indistinguishable from the signals from the [4Fe-4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [Fe-4S](2+)-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal with g approximately 2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.

Iron species in iron homeostasis and toxicity.

Iron homeostasis in prokaryotic cells appears to be regulated essentially at the level of the genome by the Fur protein. When iron is in short supply the uptake and assimilation pathways are de-repressed and siderophores are synthesized together with the outer, inner (plasma) membrane, periplasmic and cytosolic components necessary for the uptake of ferri-siderophores. When iron is no longer limiting the Fe2+ the Fur complex acts as a transcriptional repressor, and shuts down the synthesis of all the components of iron assimilation. In euykarotic cells, iron homeostasis is dependent upon the iron regulatory factor (IRF), a cytoplasmic protein that can bind to specific stem loops, iron responsive elements (IREs) on the messenger ribonucleic acid molecules (mRNAs) of proteins involved in iron storage (ferritin), utilization (erythroid delta-aminolaevulinate synthase, AIS), and uptake (transferrin receptor). During ion depletion the IRE is in a high affinity form, which, by binding strongly to the corresponding mRNAs, down regulates iron storage and utilization, while up-regulating transferrin receptor expression. When the cells are iron replete, IRF binding to IREs is weak, allowing transferrin receptor mRNA to be degraded. In this paper it is shown that in physiological conditions of iron overload and depletion, IRF functions in vivo in the manner already described for in vitro models. The nature and the speciation of the various iron species within the low molecular weight pool of eukaryotic cells remains unclear.

Duodenal ferritin synthesis in genetic hemochromatosis

Background/Aims: The molecular defect of genetic hemochromatosis (GH) is unknown. It is believed that low expression of duodenal ferritin in GH is caused by tissue or cell specific defect of ferritin synthesis. Our study was designed to ascertain whether the control of duodenal ferritin synthesis in GH was defective. Methods: Expression at the single cell level of H and L ferritin messenger RNAs and protein and activity of the iron regulatory factor, which controls the translation of ferritin messenger RNA, were assessed in 43 duodenal biopsy specimens from individuals with GH, secondary hemochromatosis (SH), anemia, or normal iron balance. Results: Signal for ferritin H and L subunit messenger RNAs was detected in both absorptive and nonabsorptive cells by in situ hybridization, but in 10 of 14 patients with untreated GH, the signal was lower than in patients with SH or normal subjects. However, immunostaining for ferritin protein documented a diffuse/cytoplasmic pattern, whereas a supranuclear/granular staining was found in normal subjects or patients with SH. The spontaneous activity of duodenal iron regulatory factor was consistently higher in patients with GH than in normal subjects or subjects with anemia or SH. Conclusions: In patients with GH, ferritin gene transcription is preserved in both absorptive and nonabsorptive intestinal cells. Low accumulation of ferritin is not caused by a defective control of ferritin synthesis but by low expression of ferritin messenger RNA and sustained activity of iron regulatory factor.