Recent data have indicated a fundamental role of iron in mediating a non-apoptotic and non-necrotic oxidative form of programmed cell death termed ferroptosis that requires abundant cytosolic free ...labile iron to promote membrane lipid peroxidation. Different scavenger molecules and detoxifying enzymes, such as glutathione (GSH) and glutathione peroxidase 4 (GPX4), have been shown to overwhelm or exacerbate ferroptosis depending on their expression magnitude. Ferroptosis is emerging as a potential weapon against tumor growth since it has been shown to potentiate cell death in some malignancies. However, this mechanism has been poorly studied in Rhabdomyosarcoma (RMS), a myogenic tumor affecting childhood and adolescence. One of the main drivers of RMS genesis is the Retrovirus Associated DNA Sequences/Extracellular signal Regulated Kinases (RAS/ERK)signaling pathway, the deliberate activation of which correlates with tumor aggressiveness and oxidative stress levels. Since recent studies have indicated that treatment with oxidative inducers can significantly halt RMS tumor progression, in this review we covered different aspects, ranging from iron metabolism in carcinogenesis and tumor growth, to mechanisms of iron-mediated cell death, to highlight the potential role of ferroptosis in counteracting RMS growth.
Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is ...considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum–Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body–exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.
•Iron-loaded ferritin is secreted via both the nonclassical secretory autophagy and multivesicular body–exosome pathways.•A motif on both ferritin subunits is involved in the regulation of ferritin secretion.
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Ferritinophagy is the process of autophagic degradation of ferritin that participates in the regulation of cellular iron homeostasis. This process was shown to be mediated by the selective ...cargo-receptor Nuclear Receptor Coactivator-4 (NCOA4) that binds ferritin and targets it to emerging autophagosome. To characterize some of the biochemical properties of the interaction between the two proteins we cloned and expressed in E. coli the ferritin-binding domain of human NCOA4, fragment 383–522. It was purified and subjected to biochemical analysis. The NCOA4(383–522) fragment was expressed in soluble and dimeric form, and CD spectra indicated low level of secondary structure. The Ferritin binding activity of the fragment was investigated by developing an electrophoretic mobility shift and an ELISA assays. They showed that the NCOA4 fragment binds the H-ferritin with an affinity in the nM range, but not the R23A H-ferritin mutant and the L-ferritin chain, confirming the high specificity for the H-chain. The H-ferritin could bind up to 24 NCOA4(383–522) fragments forming highly stable and insoluble complexes. The binding was partially inhibited only by Fe(II) among the various divalent metal ions analyzed. The iron-dependent, highly-specific formation of the remarkably stable H-ferritin-NCOA4 complex shown in this work may be important for the characterization of the mechanism of ferritinophagy.
•The recombinant ferritin binding domain of NCOA4 (residues 383-522) was expressed in E. coli and purified.•The domain binds ferritin H-chain with high affinity, but not its mutant Arg23Ala and ferritin L-chain•Electro mobility shift assay showed that H-ferritin binds up to 24 NCOA4 domains forming a highly stable complex•ELISA assay showed that the complex formation is partially inhibited by Fe(II) but not by other divalent metal ions.
In animals, the iron storage and detoxification protein, ferritin, is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light), which co-assemble in various ratios ...with tissue specific distributions to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunit possesses a ferroxidase center (FC) that catalyzes Fe(II) oxidation, whereas the L-subunit does not. To assess the role of the L-subunit in iron oxidation and core formation, two human recombinant heteropolymeric ferritins, designated H-rich and L-rich with ratios of ∼20H:4L and ∼22L:2H, respectively, were employed and compared to the human homopolymeric H-subunit ferritin (HuHF). These heteropolymeric ferritins have a composition similar to the composition of those found in hearts and brains (i.e., H-rich) and in livers and spleens (i.e., L-rich). As for HuHF, iron oxidation in H-rich ferritin was found to proceed with a 2:1 Fe(II):O2 stoichiometry at an iron level of 2 Fe(II) atoms/H-subunit with the generation of H2O2. The H2O2 reacted with additional Fe(II) in a 2:1 Fe(II):H2O2 ratio, thus avoiding the production of hydroxyl radical. A μ-1,2-peroxo-diFe(III) intermediate was observed at the FC of H-rich ferritin as for HuHF. Importantly, the H-rich protein regenerated full ferroxidase activity more rapidly than HuHF did and additionally formed larger iron cores, indicating dual roles for the L-subunit in facilitating iron turnover at the FC and in mineralization of the core. The L-rich ferritin, while also facilitating iron oxidation at the FC, additionally promoted oxidation at the mineral surface once the iron binding capacity of the FC was exceeded.
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Hepcidin peptide is crucial in the regulation of systemic iron availability controlling its uptake from the diet and its release from the body storage tissues. Hepcidin dysregulation ...causes different human disorders ranging from iron overload (e.g. hemochromatosis) to iron deficiency (e.g. anemia). Hepcidin excess is common in the Anemia of Chronic Diseases or Anemia of Inflammation and in the genetic form of anemia named IRIDA; the pharmacological downregulation of hepcidin in these disorders could improve the anemia. Commercial heparins were shown to be strong inhibitors of hepcidin expression, by interfering with BMP6/SMAD pathway. The non-anti-coagulant heparins, modified to abolish the anti-thrombin binding site, were equally potent and could be used to improve iron status. To perform its anti-hepcidin activity heparin needs 2O- and 6O-sulfation and an average molecular weight (MW) up to 4000–8000 Dalton, depending on the sulfation level. The pentosane polysulfate (PPS), which shares with heparin a high degree of sulfation, is a compound with low anti-coagulant activity that is already in use for pharmaceutical treatment. In the present work we analyzed the anti-hepcidin activity of PPS in vitro and in vivo. We found that it acts as a strong inhibitor of hepcidin expression in HepG2 cells with an effect already visible after 2–3 h of treatment. It also suppressed hepcidin in mice in a dose dependent manner after 3 h and with a significant redistribution of systemic iron without evident side effects. PPS is also able to abolish the LPS dependent hepcidin upregulation similarly to that showed for heparin derivatives. These results suggest PPS as an interesting compound to control hepcidin in vivo.
Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in ...regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to protection against lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP)-induced model of sepsis as evidenced by reduced cytokine levels, multi-organ dysfunction and mortality. We identified that such protection is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our
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studies indicate that prior exposure to ferritin light chain restrains an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of FtL on NF-κB activation, a key signaling pathway that is implicated in the pathogenesis of sepsis. We further identified that LPS mediated activation of MAPK pathways, specifically, JNK, and ERK were also reduced with FtL pre-treatment. Taken together, our findings elucidate a crucial immunomodulatory function for circulating ferritin that challenges the traditional view of this protein as a mere marker of body iron stores. Accordingly, these findings will stimulate investigations to the adaptive nature of this protein in diverse clinical settings.
The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs), which are heparin binding ...proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.
Hepcidin is a major regulator of iron homeostasis, and its expression in liver is regulated by iron, inflammation, and erythropoietic activity with mechanisms that involve bone morphogenetic proteins ...(BMPs) binding their receptors and coreceptors. Here we show that exogenous heparin strongly inhibited hepcidin expression in hepatic HepG2 cells at pharmacologic concentrations, with a mechanism that probably involves bone morphogenetic protein 6 sequestering and the blocking of SMAD signaling. Treatment of mice with pharmacologic doses of heparin inhibited liver hepcidin mRNA expression and SMAD phosphorylation, reduced spleen iron concentration, and increased serum iron. Moreover, we observed a strong reduction of serum hepcidin in 5 patients treated with heparin to prevent deep vein thrombosis, which was accompanied by an increase of serum iron and a reduction of C-reactive protein levels. The data show an unrecognized role for heparin in regulating iron homeostasis and indicate novel approaches to the treatment of iron-restricted iron deficiency anemia.
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