Anna Cozzi

Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron-deficient phenotype

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline‐repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (∼10‐fold) decrease in H‐ and L‐ferritin levels, by a ∼3–4‐fold increase in transferrin receptor, and a ∼2‐fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed ∼30% decrease in 55Fe‐transferrin incorporation after 18 h incubation. In the HFE‐expressing cells the reduction in transferrin‐mediated iron incorporation was partially compensated by a ∼30% increase in non‐transferrin iron incorporation from 55Fe‐NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin‐mediated and non‐transferrin‐mediated cellular iron incorporation.

Role of iron and ferritin in TNFα-induced apoptosis in HeLa cells

We found that tumor necrosis factor α (TNFα)‐induced apoptosis in HeLa cells was accompanied by a ∼2‐fold increase in H‐ and L‐ferritin and a decrease in transferrin receptor, two indices of increased iron availability. Iron supplementation and overexpression of H‐ferritin or its mutant with an inactivated ferroxidase center reduced by about ∼50% the number of apoptotic cells after TNFα‐treatment, while overexpression of L‐ferritin was ineffective. The data indicate that H‐ferritin has an anti‐apoptotic activity unrelated to its ferroxidase activity and to its capacity to modify cellular iron metabolism.

Human serum ferritin G-peptide is recognized by anti-L ferritin subunit antibodies and concanavalin-A

Summary. Ferritin was purified from serum of patients with idiopatic haemochromatosis. Analysis on SDS electrophoresis showed that it is composed of two major bands of 19 000 and 23000 Mr. The smaller peptide has an electrophoretic mobility and immunochemical reactivity similar to that of tissue L subunit. The larger, previously named G subunit, is recognized by concanavalin‐A and by anti ferritin L‐subunit, but not by anti‐H, monoclonal antibodies. All of the antibodies show higher affinity for the L than for the G subunit. Therefore, the G chain appears immunochemically similar, but not identical, to ferritin L chain, and is responsible for serum ferritin binding concanavalin‐A.

Oxidative stress and cell death in cells expressing L-ferritin variants causing neuroferritinopathy

Neuroferritinopathies are dominantly inherited movement disorders associated with nucleotide insertions in the L-ferritin gene that modify the proteins C-terminus. The insertions alter physical and functional properties of the ferritins, causing an imbalance in brain iron homeostasis. We describe the effects produced by the over-expression in HeLa and neuroblastoma SH-SY5Y cells of two pathogenic L-ferritin variants, 460InsA and 498InsTC. Both peptides co-assembled with endogenous ferritins, producing molecules with reduced iron incorporation capacity, acting in a dominant negative manner. The cells showed an increase in cell death and a decrease in proteasomal activity. The formation of iron-ferritin aggregates became evident after 10 days of variant expression and was not associated with increased cell death. The addition of iron chelators or antioxidants restored proteasomal activity and reduced aggregate formation. The data indicate that cellular iron imbalance and oxidative damage are primary causes of cell death, while aggregate formation is a secondary effect.

Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

Case report: a subject with a mutation in the ATG start codon of L-ferritin has no haematological or neurological symptoms

Ferritin consists of two subunit types, H and L, which assemble in different proportions in a 24-mer protein.1 The H-subunit has ferroxidase activity and is mainly found in cell cytoplasm, where it has the major function of sequestering and detoxifying unwanted iron. The L-subunit has no catalytic activity on its own, but assists the functionality of the H-subunit2 and is also found in minor amounts in serum.3 Two types of genetic disorder are associated with mutations of the L-ferritin gene ( FTL ), both with autosomal dominant transmission. The first, hereditary hyperferritinaemia cataract syndrome (HHCS), is caused by mutations in the regulatory iron responsive element (IRE) in the 5′UTR of the transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to constitutive upregulation of the protein in tissue and serum.4–8 Subjects with the mutations show high levels of serum ferritin (500–2000 μg/l) and often early-onset bilateral cataracts6 likely caused by protein aggregation in the lens,9 but do not present alterations in iron metabolism. The disorder has been extensively studied and more than 21 different causative mutations have been identified.9,10 The second type of genetic disorder, neuroferritinopathy, is rare and few families have been identified. It is associated with an adenine insertion at position 460–461 in the coding region (exon 4) of the gene that causes a frame shift alteration of the C-terminus of the L-ferritin polypeptide.11 Affected subjects show late-onset movement disorders, iron deposition in the brain basal ganglia, and low serum ferritin levels.12,13 It is unclear whether the iron deposition in the brain is caused by a quantitative defect of L-ferritin or by an abnormal functionality caused by …

Development of an immunoassay for all human isoferritins, and its application to serum ferritin evaluation

Calibrated mixtures of anti-H and anti-L ferritin subunit monoclonal antibodies were used in a sandwich enzyme-immunoassay for the evaluation of all isoferritins. The assay was designed to have overlapping calibration plots for human liver (95% L-chain) and recombinant human H-chain ferritin (100% H-chain). It appeared to recognize all the heart isoferritins including the ones in the middle of the isoferritin spectrum. By direct comparison it was shown that these isoferritins are under-evaluated by the assays for H- and L-subunit-rich ferritins, based on the two separated antibodies. The three assays (for total, H-rich and L-rich ferritins) provide an index of the presence of the isoferritins with intermediate H/L composition. Sera from 30 tumor and non-tumor patients were analyzed. Intermediate isoferritins were found in 2 non-tumor subjects and in none of the 14 patients with Hodgkins disease or mammary carcinoma. It is concluded that the evaluation of the total and intermediate isoferritins is possible, but does not have an evident clinical significance for tumor monitoring.

Mutant ferritin L-chains that cause neurodegeneration act in a dominant-negative manner to reduce ferritin iron incorporation.

Nucleotide insertions that modify the C terminus of ferritin light chain (FTL) cause neurodegenerative movement disorders named neuroferritinopathies, which are inherited with dominant transmission. The disorders are characterized by abnormal brain iron accumulation. Here we describe the biochemical and crystallographic characterization of pathogenic FTL mutant p.Phe167SerfsX26 showing that it is a functional ferritin with an altered conformation of the C terminus. Moreover we analyze functional and stability properties of ferritin heteropolymers made of 20–23 H-chains and 1–4 L-chains with representative pathogenic mutations or the last 10–28 residues truncated. All the heteropolymers containing the pathogenic or truncated mutants had a strongly reduced capacity to incorporate iron, both when expressed in Escherichia coli, and in vitro when iron was supplied as Fe(III) in the presence of ascorbate. The mutations also reduced the physical stability of the heteropolymers. The data indicate that even a few mutated L-chains are sufficient to alter the permeability of 1–2 of the 6 hydrophobic channels and modify ferritin capacity to incorporate iron. The dominant-negative action of the mutations explains the dominant transmission of the disorder. The data support the hypothesis that hereditary ferritinopathies are due to alterations of ferritin functionality and provide new input on the mechanism of the function of isoferritins.

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

Human L-ferritin deficiency causes reduced cellular iron availability and increased ROS production with enhanced oxidized proteins, which results in idiopathic generalized seizures and atypical restless leg syndrome.

Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

Background Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. Design and Methods We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Results Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Conclusions Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation.