Role In Iron Metabolism Research Articles

Structural and Functional Diversity of Glutaredoxins in Yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.

Hydroxyurea Induces Hepcidin Expression In Monocytes In Sickle Cell Anemia Patients

AbstractAbstract 2653The antimicrobial peptide hormone hepcidin is a key regulator of iron metabolism. Although mainly produced in the liver, hepcidin is also known to be synthesized by monocytic-macrophagic cells. We have previously shown that hepcidin is overexpressed in mononuclear cells in patients with sickle cell anemia (SCA), but whether monocyte-derived hepcidin production is related to iron metabolism remains unknown.To gather further insight into the role of hepcidin in monocytes, we collected peripheral blood samples from adult SCA patients (n=54) and normal age- and race-matched controls (CTRL, n=13) for assessment of hematological parameters, biochemical markers of iron status (serum iron and transferrin saturation - TfSat), hemolysis (lactate dehydrogenase-LDH) and inflammation (C reactive protein - CRP), and separation of peripheral blood monocytes by Ficoll-Hypaque and Percoll gradients for gene expression analyses of genes involved in iron metabolism signaling (HAMP encoding hepcidin, and three genes belonging to different pathways known to influence hepcidin expression, STAT3, SMAD4 and TLR4). Plasma GDF-15 levels were also measured, as this protein has been shown to be a potent downregulator of hepcidin. SCA patients were further stratified according to the number of previous blood transfusions and to treatment with hydroxyurea (HU). All patients were in steady-state, had no history of iron chelation treatment and were not enrolled in a regular transfusion program. 18 patients were receiving HU and 15 from the non-HU group had received over 20 blood transfusions during their lifetime.As expected, SCA patients had elevated LDH levels, but no differences were found between control and SCA groups regarding serum iron, TfSat or CRP levels. Except for a higher red cell mean corpuscular volume, patients on HU did not differ significantly from patients not using HU. Plasma GDF-15 levels were higher in SCA patients (2146±506.4pg/mL) than in control individuals (228.5±21.0pg/mL, p<0.0001). Among the genes studied, HAMP expression was significantly increased in the SCA group as a whole compared to the CTRL group, but SCA patients on HU had higher levels of monocytic hepcidin expression when compared to the remaining individuals (CTRL 0.043±0.030, SCA on HU 1.240±0.426, remaining SCA 0.332±0.093, p=0.0196). There were no significant correlations between monocytic hepcidin expression and hemoglobin levels, hematocrit, leucocyte or reticulocyte counts, serum iron, LDH or CRP levels, TfSat or transfusion history. STAT3, SMAD4 and TLR4 gene expressions did not differ significantly, suggesting that a possible alternative cause for hepcidin upregulation unrelated to known mechanisms by IL-6, BMP6, LPS or GDF-15 could be an unexpected effect of hydroxyurea. To further investigate if HU was able to modulate hepcidin expression, we performed experiments with THP-1, a human monocytic lineage, since in vitro analysis would allow us to exclude the influence of circulating cytokines elevated in SCA patients. THP-1 cells were cultured in RPMI medium enriched with 10% fetal bovine serum at 37°C and 5% CO2 atmosphere, and were submitted to treatment with water as control, or HU dissolved in water in increasing concentrations (100μM, 400μM and 1600μM) for 6 hours (n≥4). Cell viability was not affected by treatment (>90% viable cells at all experiments), and HAMP gene expression was increased up to four times in the cell cultures exposed to HU (p=0.03), while STAT3 and SMAD4 expressions remained unchanged.We have shown that hepcidin expression is upregulated in monocytes in SCA patients, particularly in those receiving HU, and that HU is capable of inducing this expression in an in vitro model, independently from inflammatory cytokine-mediated stimulation. Our data suggest that, although liver-derived hepcidin has been shown to have a major role in iron metabolism, its monocyte-derived counterpart does not seem to be directly influenced by iron status and may have other functions. Some studies have demonstrated that hepcidin in other species has anti-inflammatory effects in vitro, and that patients with SCA on HU shift to a lower inflammatory status. Thus, monocytic hepcidin overexpression might be a response against the chronic inflammatory state in SCA, and HU treatment may enhance this response. This is the first description of monocyte-derived hepcidin in SCA and the influence of HU on its expression. Disclosures:No relevant conflicts of interest to declare.

In silico QTL mapping of basal liver iron levels in inbred mouse strains

Both iron deficiency and iron excess are detrimental in many organisms, and previous studies in both mice and humans suggest that genetic variation may influence iron status in mammals. However, these genetic factors are not well defined. To address this issue, we measured basal liver iron levels in 18 inbred strains of mice of both sexes on a defined iron diet and found ∼4-fold variation in liver iron in males (lowest 153 μg/g, highest 661 μg/g) and ∼3-fold variation in females (lowest 222 μg/g, highest 658 μg/g). We carried out a genome-wide association mapping to identify haplotypes underlying differences in liver iron and three other related traits (copper and zinc liver levels, and plasma diferric transferrin levels) in a subset of 14 inbred strains for which genotype information was available. We identified two putative quantitative trait loci (QTL) that contain genes with a known role in iron metabolism: Eif2ak1 and Igf2r. We also identified four putative QTL that reside in previously identified iron-related QTL and 22 novel putative QTL. The most promising putative QTL include a 0.22 Mb region on Chromosome 7 and a 0.32 Mb region on Chromosome 11 that both contain only one candidate gene, Adam12 and Gria1, respectively. Identified putative QTL are good candidates for further refinement and subsequent functional studies.

Conserved proximal promoter elements control repulsive guidance molecule c/hemojuvelin (Hfe2) gene transcription in skeletal muscle

Repulsive guidance molecule c (RGMc; gene symbol: Hfe2) plays a critical role in iron metabolism. Inactivating mutations cause juvenile hemochromatosis, a severe iron overload disorder. Understanding mechanisms controlling RGMc biosynthesis has been hampered by minimal information about the RGMc gene. Here we define the structure, examine the evolution, and establish mechanisms of regulation of the mouse RGMc gene. RGMc is a 4-exon gene that undergoes alternative RNA splicing to yield 3 mRNAs with 5′ different untranslated regions. Gene transcription is induced during myoblast differentiation, producing all 3 mRNAs. We identify 3 critical promoter elements responsible for transcriptional activation in skeletal muscle, comprising paired E-boxes, a putative Stat and/or Ets element, and a MEF2 site, and muscle transcription factors myogenin and MEF2C stimulate RGMc promoter function in non-muscle cells. As these elements are conserved in RGMc genes from multiple species, our results suggest that RGMc has been a muscle-enriched gene throughout its evolutionary history.

Deciphering the iron response in Acinetobacter baumannii: A proteomics approach

Iron is an essential nutrient that plays a role in bacterial differential gene expression and protein production. Accordingly, the comparative analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606(T) cells cultured under iron-rich and -chelated conditions using 2-D gel electrophoresis-mass spectrometry resulted in the identification of 58 protein spots differentially produced. While 19 and 35 of them represent iron-repressed and iron-induced protein spots, respectively, four other spots represent a metal chelation response unrelated to iron. Most of the iron-repressed protein spots represent outer membrane siderophore receptors, some of which could be involved in the utilization of siderophores produced by other bacteria. The iron-induced protein spots represent a wide range of proteins including those involved in iron storage, such as Bfr, metabolic and energy processes, such as AcnA, AcnB, GlyA, SdhA, and SodB, as well as lipid biosynthesis. The detection of an iron-regulated Hfq ortholog indicates that iron regulation in this bacterium could be mediated by Fur and small RNAs as described in other bacteria. The iron-induced production of OmpA suggests this protein plays a role in iron metabolism as shown by the diminished ability of an isogenic OmpA deficient derivative to grow under iron-chelated conditions.

Hepicidin and its role in iron metabolism

Among various micro-nutrients, iron plays a major role not only for hemoglobin alone but also for oxidative metabolism and energy production. Hemoglobin functions as a chief oxygen carrier while other iron containing mitochondrial enzymes and respiratory chain proteins are involved in oxidative metabolism and release of energy from carbohydrates and fats. Iron salts have very low bioavailability and have potential for toxicity. Therefore iron absorption and its metabolism in humans is highly regulated process. Its regulation at cellular levels is through iron responsive proteins via ironresponsive elements in messenger RNA from genes encoding proteins of iron metabolism. Regulation of iron metabolism along with iron mobilization from macrophages, tissue stores and synthesis of transferrin receptor and ferritin is a complex process. It has been observed that hepatocytes produce hormone peptide hepcidin which plays a major role in the systemic regulation of iron metabolism by its action on target cells at various sites including the liver. Zhang and Enns have recently concluded that hepicidin plays a key of role in the regulation of iron metabolism.1 Several experimental and other clinical studies have demonstrated that hepicidin controls the release of iron from variety of cells such as macrophages, hepatocytes, enterocytes etc to plasma.2-5 Hepcidin primarily controls the iron absorption. However, the recycling of iron from red cell lysis and release of iron from tissue iron stores is carried by the interaction of hepicidin with ferroportin which is a cellular iron exporter in vertebrates.5 Ferroportin is primarily expressed by enterocytes and macrophages. It has been shown that release of ferroportin is also controlled by hepicidin.

Molecular characterization and gene expression of the channel catfish ferritin H subunit after bacterial infection and iron treatment

Ferritins are the major iron storage protein in the cytoplasm of cells, responsible for regulating levels of intracellular iron. Ferritin genes are widely distributed in both prokaryotes and eukaryotes. In mammals, ferritin molecules are composed of heavy- (H) and light- (L) chain subunits; amphibian genomes contain three ferritin-type genes (H; middle, M; and L subunits); and teleost genomes to date contain H and M subunits. The objective of this study was to characterize the ferritin H gene in channel catfish (Ictalurus punctatus) to determine its genomic organization and copy numbers, to determine its patterns of tissue expression, and to establish if it is involved in defense responses of catfish after bacterial infection. The catfish ferritin H gene was completely sequenced and characterized, using both mRNA and genomic DNA. Catfish ferritin H gene has a full-length mRNA sequence of 999 bp, an open reading frame of 534 bp, and 4,704 bp genomic DNA sequence. Catfish ferritin H has a 5 exon and 4 intron genetic organization, containing a long 5'-untranslated region, which shares high similarity with mammalian and zebrafish genes. Based on phylogenetic analyses, the catfish ferritin H gene is highly conserved throughout evolution. Southern blot analysis suggested that the ferritin H gene has only one copy in the catfish genome. The catfish ferritin H gene was widely expressed in various healthy tissues. The catfish ferritin H gene was significantly up-regulated in the liver after intraperitoneal injection of iron dextran and coinjection of Edwardsiella ictaluri and iron dextran treatment, suggesting its role in iron metabolism and immunity.

Proteomic Analysis of Plasma in the Polycystic Ovary Syndrome Identifies Novel Markers Involved in Iron Metabolism, Acute-Phase Response, and Inflammation

Hypothesis-free approaches such as proteomic analysis may identify novel biomarkers for disease. The objective of the study was to compare the plasma proteome of patients presenting with the polycystic ovary syndrome (PCOS) with that of women without hyperandrogenism. This was a case-control study. The study was conducted at an academic hospital. Patients included 12 PCOS patients and 12 women without hyperandrogenism. Interventions included basal blood sampling. Two-dimensional difference in-gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Western blot, and ELISA analyses were measured. Two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analyses identified haptoglobin beta-chain and alpha2-macroglobulin as proteins underexpressed in PCOS samples, whereas transferrin and kappa-free light chain were overexpressed. We were able to confirm only the underexpression of haptoglobin beta-chain in subsequent Western blot and ELISA analyses. Proteomic analysis of plasma from PCOS patients revealed changes in protein expression in several acute-phase response proteins including isoforms of plasma haptoglobin, alpha2-macroglobulin, and transferrin and in kappa-free light chain. In addition to their role as inflammatory markers, some of these molecules play major roles in iron metabolism, further suggesting that iron metabolism and low-grade chronic inflammation may be involved in the pathogenesis of insulin-resistant disorders such as PCOS.

Abstract 1293: Neutrophil gelatinase-associated lipocalin regulates the expression of angiogenic cytokines and VEGF in human breast and ovarian cancer cells

Abstract Neutrophil gelatinase-associated lipocalin (NGAL; lipocalin 2) is a small extracellular protein (25 kDa) that is a member of the lipocalin protein family. It has been shown to bind ferric siderophores and to play an important role in iron metabolism. Increased NGAL levels have been reported in a variety of epithelial cancers, including breast, ovary, pancreas, colon, esophagus and thyroid cancer. We have recently reported that NGAL promotes breast cancer progression by inducing the epithelial to mesenchymal transition. In the course of these studies, we observed an increase in microvessel density in NGAL-expressing tumors. In the current study, we explored the potential role of NGAL in tumor angiogenesis by studying its effects on the expression of angiogenic factors. We first determined the levels of vascular endothelial growth factor (VEGF) in NGAL-expressing cells derived from the MCF-7 human breast cancer cell line. Using reverse-transcriptase PCR and ELISA, VEGF was found to be induced by NGAL at both the transcript and protein levels. Consistently, HIF-1α, one of the most important transcriptional regulators of VEGF, was observed to be accumulated in the nuclei of NGAL-expressing cells. Silencing of HIF-1α in NGAL-expressing cells significantly reduced VEGF expression. Consistent with this NGAL-induced VEGF production, there was a significant increase in endothelial cell migration towards the conditioned media collected from NGAL-expressing cells. Interestingly, treatment with a VEGF-neutralizing antibody only partially inhibited the migration-inducing activity of the conditioned media, suggesting that NGAL might induce the secretion of other angiogenic factors. To investigate this possibility, a panel of angiogenic factors was compared between the conditioned media collected from NGAL-expressing cells and control MCF-7 cells using antibody arrays. Several angiogenic cytokines, including IL-6, MCP-1 and GM-CSF, were found to be upregulated with NGAL expression. The effects of NGAL on VEGF and angiogenic cytokine production were also studied in SKOV-3 ovarian cancer cells. In this study, NGAL was silenced in SKOV-3 cells using specific siRNAs. The levels of both VEGF and IL-6 were found to be reduced after NGAL silencing, suggesting that NGAL's control of VEGF and cytokine production may be common among cancer cell types. These results suggest a novel mechanism through which NGAL may promote tumor angiogenesis and progression. (This work is supported by NIH R01 CA118764, P01 CA045548 and The Advanced Medical Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1293.

Native and Synthetic Ferritins for Nanobiomedical Applications: Recent Advances and New Perspectives

Ferritin is the protein whose function is to store iron that the cell does not require immediately for metabolic processes, thereby protecting against the toxic effects of free Fe(2+). Ferritin therefore plays a crucial role in iron metabolism as well as in the development of some diseases, especially those related to the presence of free Fe(2+) and toxic hydroxyl radicals. In addition, ferritin is itself a catalytic bionanoparticle. Its internal cavity can be used as a nanoreactor to produce non-native metallic nanoparticles. Moreover, its external protein shell can be chemically modified, allowing ferritin to be used as a precursor for a library of metallic nanoparticles, some which may have potential applications in biomedicine, especially as multimodal imaging probes. This article presents a brief overview of the evidence for the role of native ferritin in some diseases, as well as the potential of some synthetic ferritins--in which a non-native inorganic material has been introduced into the cavity and/or the external shell has been modified--in the field of nanobiomedicine.

Analysis of the High-Affinity Iron Uptake System at the Chlamydomonas reinhardtii Plasma Membrane

Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe(3+) transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonas reinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. We show that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidase domain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S. cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeation motifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1 and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator does not reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity iron uptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase.

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

The liver and the spleen are the organs in which cellular material and aged erythrocytes are eliminated from the blood. Within the liver, Kupffer cells (KCs) are mainly responsible for this task, as such KCs have a pivotal role in iron metabolism. The aim of this study is to investigate the changes of hepatic gene expression in two models of KC phagocytosis. Gadolinium chloride (GD) or zymosan was injected intraperitoneally into rats and to endotoxin-resistant mice (C3H/HeJ). The animals were killed at different time points and their livers were immediately frozen in liquid nitrogen for RNA isolation and immunohistological studies. RNA was analyzed by real-time PCR and northern blot. Sera were used to measure transaminases, hepcidin and iron levels. The expression of iron metabolism genes, hepcidin, hemojuvelin (Hjv), ferroportin-1 (Fpn-1) and of the inflammatory cytokines IL-6, IL-1β, TNF-α and IFN-γ was determined. Although phagocytosed material was detected in ED-1- and C1q-positive cells, no inflammatory cells were identified within the liver parenchyma. Serum levels of hepcidin, iron and transaminases did not differ from those of control animals. Both GD and zymosan induced an upregulation of hepcidin-gene expression in rat liver as early as 3 h, reaching a maximum 6 h after treatment. Hjv- and Fpn-1-gene expression was downregulated at the same time. IL-6 was by far the most induced acute-phase-cytokine in GD- and zymosan-treated livers, although IL-1β and TNF-α were also strongly upregulated by zymosan and to a lesser extent by GD. Similar results were obtained in the C3H/HeJ mouse strain excluding the possible role of contaminating endotoxin. This study shows that phagocytosis upregulates hepcidin-gene expression and downregulates Hjv- and Fpn-1-gene expression within the liver. These changes in iron-regulating-gene expression may be mediated by the locally produced acute-phase-cytokines.

Mutations in the Gene Encoding DMT1: Clinical Presentation and Treatment

Divalent metal transporter 1 (DMT1) is the protein that allows elemental iron entry into the duodenal cell. It is expressed ubiquitously and it also allows the iron exit from the endosomes. This protein plays a central role in iron metabolism and it is strictly regulated. Several animal models elucidate its role in physiology. Recently three patients affected with DMT1 deficiency have been described. This recessively inherited condition appears at birth with severe microcytic anemia. Serum markers could be particularly useful to establish a correct diagnosis: high serum iron, normal total iron-binding capacity (TIBC), increased saturation of transferrin (Tf), slightly elevated ferritin, and increased soluble transferrin receptor (sTfR). Increased free erythrocyte protoporphyrins (FEPs) could address the diagnosis to iron-deficient anemia. All patients appeared to respond to erythropoietin (Epo) administration. Because mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) did not change during Epo treatment, it was concluded that Epo did not improve iron utilization of the erythroblasts but likely reduced the degree or intensity of apoptosis, affecting erythropoiesis. Moreover liver iron overload was present and documented in all of the affected patients. In this review we analyze the role of DMT1 in iron metabolism and the major causes of reduction and their consequences in animal models as well in humans, and we attempt to define the correct treatment for human mutants.

Association of hepcidin promoter c.-582 A&gt;G variant and iron overload in thalassemia major

Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter.

Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma

BackgroundHepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC).MethodsWe measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction.ResultsThe serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival.ConclusionOur findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential.

HFE gene mutations and Wilson's disease in Sardinia

Hypocaeruloplasminaemia can lead to tissue iron storage in Wilson's disease and the possibility of iron overload in long-term overtreated patients should be considered. The HFE gene encodes a protein that is intimately involved in intestinal iron absorption. The aim of this study was to determine the prevalence of the HFE gene mutation, its role in iron metabolism of Wilson's disease patients and the interplay of therapy in copper and iron homeostasis. The records of 32 patients with Wilson's disease were reviewed for iron and copper indices, HFE gene mutations and liver biopsy. Twenty-six patients were negative for HFE gene mutations and did not present significant alterations of iron metabolism. The HFE mutation was significantly associated with increased hepatic iron content (P<0.02) and transferrin saturation index (P<0.03). After treatment period, iron indices were significantly decreased only in HFE gene wild-type. The HFE gene mutations may be an addictional factor in iron overload in Wilson's disease. Our results showed that an adjustment of dosage of drugs could prevent further iron overload induced by overtreatment only in patients HFE wild-type.

NGAL: a biomarker of acute kidney injury and other systemic conditions

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25 kDa protein belonging to the lipocalin superfamily. It was initially found in activated neutrophils, however, many other cells, like kidney tubular cells, may produce NGAL in response to various insults. Recently, it has been found to have a role in iron metabolism by virtue of its binding with siderophores. It has also been found to have a role in kidney development and tubular regeneration after injury. In experimental studies, it was found to be highly expressed in response to tubular injury. In subsequent clinical studies, urine NGAL has been found to be an early predictor for acute kidney injury (AKI). Newer devices for early bedside detection of NGAL are now available. Since serum creatinine is known to be an inadequate and late marker of AKI, NGAL might soon emerge as a troponin-like early marker for AKI. Recent evidence also suggests its role as a biomarker in a variety of other renal and non-renal conditions.

Production of recombinant protein and polyclonal mouse antiserum for ferritin from Sipuncula Phascolosoma esculenta

The iron storage protein, ferritin, plays a key role in iron metabolism, but its regulation and functions in many invertebrate species are still largely unknown. In our previous work, an inducible ferritin cDNA from Phascolosoma esculenta with a full-length of 1017 bp has been cloned. In this follow-up study, the deducted ferritin protein sequence was predicted to be a polypeptide of 175 amino acids with a molecular mass of 20.1955 kDa and an isoelectric point of 5.08. The cDNA sequence of P. esculenta ferritin was constructed into pET system expression system and efficiently expressed in E. coli BL21 under IPTG induction. The recombinant ferritin was detected as a 24 kDa protein by SDS-PAGE. After purification directly from the gel, the recombinant ferritin was used to immunize mice and the anti-serum was prepared. The antibody displayed a strong immunological reactivity and specificity when used in Western-blot analysis. For the first time, our work provided a set of molecular tools essential for the further studies of ferritin protein functions in P. esculenta.

Conditional deletion of ferritin H in mice induces loss of iron storage and liver damage #

Ferritin plays a central role in iron metabolism by acting both as iron storage and a detoxifying protein. We generated a ferritin H allele with loxP sites and studied the conditional ferritin H deletion in adult mice. Ten days after Mx-Cre induced deletion, ferritin H messenger RNA (mRNA) was below 5% in the liver, spleen, and bone marrow of deleted mice compared to control littermates. Mice lost their cellular iron stores indicating the requirement of ferritin H in iron deposition. Serum iron and transferrin saturation were slightly increased and correlated with a two-fold increased liver hepcidin 1 mRNA and a reduced duodenal DcytB mRNA level. Under a normal iron regimen, deleted mice survived for 2 years without visible disadvantage. Mice fed on a high iron diet prior to ferritin H deletion suffered from severe liver damage. Similarly, ferritin H deleted mouse embryonic fibroblasts showed rapid cell death after exposure to iron salt in the medium. This was reversed by wild-type ferritin H but not by a ferritin H mutant lacking ferroxidase activity. Cell death was preceded by an increase in cytoplasmic free iron, reactive oxygen species, and mitochondrial depolarization. Our results provide evidence that the iron storage function of ferritin plays a major role in preventing iron-mediated cell and tissue damage.

Novel insights into iron metabolism by integrating deletome and transcriptome analysis in an iron deficiency model of the yeast Saccharomyces cerevisiae

BackgroundIron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism.ResultsUsing functional profiling, we identified several genes known to be involved in high affinity iron uptake, in addition to novel genes that may play a role in iron metabolism. Our results provide support for the primary involvement in iron homeostasis of vacuolar and endosomal compartments, as well as vesicular transport to and from these compartments. We also observed an unexpected importance of the peroxisome for growth in iron-limited media. Although these components were essential for growth in low-iron conditions, most of them were not differentially-expressed. Genes with altered expression in iron deficiency were mainly associated with iron uptake and transport mechanisms, with little overlap with those that were functionally required. To better understand this relationship, we used expression-profiling of selected mutants that exhibited slow growth in iron-deficient conditions, and as a result, obtained additional insight into the roles of CTI6, DAP1, MRS4 and YHR045W in iron metabolism.ConclusionComparison between functional and gene expression data in iron deficiency highlighted the complementary utility of these two approaches to identify important functional components. This should be taken into consideration when designing and analyzing data from these type of studies. We used this and other published data to develop a molecular interaction network of iron metabolism in yeast.