Studies on iron metabolism in Neurospora crassa

Neurospora crassa Em 5297a secretes an ironbinding compound (X) when grown under conditions of iron deficiency. Decreasing the concentration of iron in the medium results in an increase of X and a corresponding fall in catalase activity. Under iron-deficient conditions the production of X precedes the fall in catalase activity. The iron complex of the iron-binding compound (XFe) can act as a good iron source to the organism to maintain normal growth and catalase activity, even though the iron is held very firmly in the chemical sense. While ferrichrome is as potent as XFe, as an iron source to N. crassa, ferrichrome A and ferric acethydroxamate are only partially beneficial. XFe, when provided as the sole iron source, also influences nonheme iron enzyme activities like succinic dehydrogenase and aconitase. XFe is permeable to N. crassa mycelia and is incorporated at a much faster rate compared with that from a simple chelate such as ferric citrate.

Retinoic Acid and Iron Metabolism: A Step Towards Design of a Novel Antitubercular Drug

The scenario of tuberculosis has gone deadly due to its high prevalence and emergence of widespread drug resistance. It is now high time to develop novel antimycobacterial strategies and to understand novel mechanisms of existing antimycobacterial compounds so that we are equipped with newer tuberculosis controlling molecules in the days to come. Iron has proven to be essential for pathogenesis of tuberculosis and retinoic acid is known to influence the iron metabolism pathway. Retenoic acid is also known to exhibit antitubercular effect in in vivo system. Therefore there is every possibility that retinoic acid by affecting the iron metabolism pathway exhibits its antimycobacterial effect. These aspects are reviewed in the present manuscript for understanding the antimycobacterial role of retinoic acid in the context of iron metabolism and other immunological aspects.

The functional link between HFE and hepcidin- its contribution to iron metabolism regulation

Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2013

A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism

The C282Y and H63D mutations in the HFE gene are important causes of hemochromatosis. In the elderly, these mutations might be associated with increased morbidity because of the lifelong accumulation of iron. In a population-based sample of the elderly, we determined the value of genotyping for HFE mutations to screen for subclinical hemochromatosis. HFE genotype frequencies were determined in a random group of 2095 subjects (55 years and over). In this random group, we selected within the six genotype groups a total of 342 individuals and measured their serum transferrin saturation, iron and ferritin levels. We also estimated the heritability and parameters needed to evaluate screening, including the sensitivity, specificity, positive and negative predictive values (PPV, NPV) of HFE genotypes. Iron parameters were significantly increased in subjects homozygous, heterozygous or compound heterozygous. The effect of the mutations was more pronounced in men than in women. For the H63D mutation, an allele dose effect was observed. The HFE gene explained about 5% of the variability in serum iron indices. The PPV for hemochromatosis for the C282Y homozygous was 100% in men and 67% in women. The NPV of the wild-type allele was 97% for both men and women. The sensitivity of both mutations was 70% for men and 52% for women and the specificity was 62% for men and 64% for women. Our study shows that the HFE C282Y and H63D are determinants of iron parameters in the elderly and will be effective in detecting individuals at high risk of hemochromatosis. However, when screening based on these two mutations, some individuals with subclinical hemochromatosis will be missed.

Nanocapsules loaded with iron-saturated bovine lactoferrin have antimicrobial therapeutic potential and maintain calcium, zinc and iron metabolism

Aim: This study aimed to evaluate the potential antimicrobial efficacy of alginate gel-encapsulated ceramic nanocarriers loaded with iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers/nanocapsules (AEC-CP-Fe-bLf NCs). Materials & methods: The antimicrobial activities of non-nanoformulated apo (iron free), Fe-bLf and native forms of Australian bLf against pathogenic Salmonella typhimurium (wild strain) were studied in vitro. The efficacy of AEC-CP-Fe-bLf NCs were checked in vivo using Balb/c mice model. Results: The study revealed that native bLf is more effective in combating infection than the conventional drug ciprofloxacin (0.4 mg/ml). The efficacy of the drug was also revealed in vivo when BALB/c mice that, after being challenged with S. typhimurium (200 μl of 10(8) CFU/ml suspension), were fed orally with a nanoformulated bLf diet and the infection was observed to be eliminated. However, chronic infection developed in the group of infected mice that did not receive any drug treatment, as well as the mice treated with ciprofloxacin. The immune response to bacterial infection and to various drug treatments thereafter was studied in the mice. Conclusion: The study concludes that bLf and nanoformulated Fe-bLf are more effective in the treatment of Salmonella-infected mice than ciprofloxacin.

Hemoglobin, iron metabolism and angiographic coronary artery disease (The Ludwigshafen Risk and Cardiovascular Health Study).

BACKGROUND Anemia has been shown to be a risk factor for coronary artery disease and mortality. The involvement of body iron stores in the development of CAD remains controversial. So far, studies that examined hemoglobin and parameters of iron metabolism simultaneously do not exist. METHODS AND RESULTS Hemoglobin and iron status were determined in 1480 patients with stable angiographic coronary artery disease (CAD) and in 682 individuals in whom CAD had been ruled out by angiography. The multivariate adjusted odds ratios (OR) for CAD in the lowest quartiles of hemoglobin and iron were 1.62 (95%CI: 1.22-2.16), and 2.05 (95%CI: 1.51-2.78), respectively compared to their highest gender-specific quartiles. The fully adjusted ORs for CAD in the lowest quartiles of transferrin saturation, ferritin (F) and soluble transferrin receptor (sTfR)/log10F index were 1.69 (95%CI: 1.25-2.27), 1.98 (95%CI: 1.48-2.65), and 1.64 (95%CI: 1.23-2.18), respectively compared to their highest gender-specific quartiles. When adjusting in addition for iron and ferritin the OR for CAD in the lowest quartiles of hemoglobin was still 1.40 (95%CI: 1.04-1.90) compared to the highest gender-specific quartiles. Thus, the associations between either iron status or low hemoglobin and CAD appeared independent from each other. The sTfR was only marginally associated with angiographic CAD. CONCLUSIONS Both low hemoglobin and iron depletion are independently associated with angiographic CAD.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Experimental hemochromatosis due to MHC class I HFE deficiency: Immune status and iron metabolism

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE α1 and α2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism.

Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.

Aceruloplasminemia: molecular characterization of this disorder of iron metabolism.

Ceruloplasmin is an abundant alpha 2-serum glycoprotein that contains 95% of the copper found in the plasma of vertebrate species. We report here on the identification of a genetic defect in the ceruloplasmin gene in a patient previously noted to have a total absence of circulating serum ceruloplasmin in association with late-onset retinal and basal ganglia degeneration. In this patient T2 (transverse relaxation time)-weighted magnetic resonance imaging of the brain revealed basal ganglia densities consistent with iron deposition, and liver biopsy confirmed the presence of excess iron. Although Southern blot analysis of the patient's DNA was normal, PCR amplification of 18 of the 19 exons composing the human ceruloplasmin gene revealed a distinct size difference in exon 7. DNA sequence analysis of this exon revealed a 5-bp insertion at amino acid 410, resulting in a frame-shift mutation and a truncated open reading frame. The validity of this mutation was confirmed by analysis of DNA from the patient's daughter, which revealed heterozygosity for this same 5-bp insertion. The presence of this mutation in conjunction with the clinical and pathologic findings demonstrates an essential role for ceruloplasmin in human biology and identifies aceruloplasminemia as an autosomal recessive disorder of iron metabolism. These findings support previous studies that identified ceruloplasmin as a ferroxidase and are remarkably consistent with recent studies on the essential role of a homologous copper oxidase in iron metabolism in yeast. The clinical and laboratory findings suggest that additional patients with movement disorders and nonclassical Wilson disease should be examined for ceruloplasmin gene mutations.

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Iron metabolism: a double-edged sword in the resistance of glioblastoma to therapies

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