Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome

Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.     

Autophagy,ferritin and iron chelation

Ferritin is an iron storage molecule in vertebrates that stores iron in a redox inactive form. Ferritin is synthesized in response to high cellular iron levels and is degraded and iron released when iron demand is increased. Previously we determined that the turnover of ferritin occurs via the proteasome when the iron exporter ferroportin is expressed, and via the lysosome when the iron chelator deferoxamine is given to cells. Deferoxamine is used to treat hemochromatosis, a disease of iron accumulation that can be either genetic or acquired.

Autophagy provides a mechanism by which cytosolic proteins gain access to the lumen of lysosomes. Our results suggest that entry of ferritin into lysosomes is highly specific and not a consequence of generalized engulfment of cytosolic compartments by lysosomes. Entry of ferritin is also independent of the presence of LAMP-2, which suggests that ferritin entry does not result from chaperone-mediated autophagy. In summary, in this study we identify a new route that links ferritin degradation to activation of autophagy. The identification of this pathway will help to understand the molecular events that lead to activation of deferoxamine-mediated ferritin degradation and may contribute to the design of new therapeutic strategies for iron chelation therapy.     

Synthesis of ferritin by neuroblastoma

Ferritin is an iron-containing protein which is a normal component of serum. The levels of ferritin are increased in the sera of some children with neuroblastoma, and this increase appears to be a potent indicator of prognosis. To determine whether synthesis of ferritin by the tumor cells contributes to these increased serum levels, we examined incorporation of radiolabeled leucine by CHP 126, a neuroblastoma derived cell line, into ferritin. Using sequential immunoprecipitation and gel electrophoresis of sonicates from cells maintained in medium containing iron in amounts standard for tissue culture, incorporation of label into ferritin was 0.04% of that into total protein synthesized over the same time period. Addition of up to 40 micrograms of iron as ferric ammonium citrate increased ferritin synthesis to a maximum of 0.16% without altering synthesis of total protein. The pattern of iron-induced enhancement in the neuroblastoma cells was similar to that which was seen using Chang liver cells, a cell line well known to be capable of ferritin synthesis. These results confirm that neuroblastoma cells can synthesize ferritin and that synthesis is regulated by exogenous iron.     

Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

Abscisic acid and oxidative stress implications in overall ferritin synthesis by African rice (Oryza glaberrima Steud.) seedlings exposed to short term iron toxicity

Iron toxicity occurs under flooded conditions such as those prevailing in lowland rice fields and is due to an excess of ferrous ions. Ferritin is a multimeric protein responsible for Fe sequestration and storage, playing a key role in Fe homeostasis. Our aim was to study the modalities of overall ferritin synthesis in different organs of young seedlings from the African rice species (Oryza glaberrima) in relation to the putative involvement of abscisic acid (ABA) and oxidative stress in signalling processes. Seedlings from a moderately resistant to iron toxicity cultivar were grown in hydroponic culture for 2 weeks and treated with 500 mg l^?1 Fe²⁺ in the presence or in the absence of 200?µM ABA, 50?µM methylviologen or 50?µM fluridone. Iron treatment increased iron and malondialdehyde concentration in all organs as well as ABA in roots and laminae. Although ferritin protein was detected in controls plants, iron treatment strongly reinforced its accumulation in sheaths and laminae after 24 h and 72 h. Ferritin mRNA was induced as early as 24 h after the beginning of the Fe-treatment in sheaths and, to a higher extent, in laminae. In the absence of iron treatment, exogenous ABA increased ferritin mRNA in laminae only but did not lead to further ferritin accumulation. Unexpectedly, it decreased ferritin mRNA levels in the sheaths of iron-treated plants and may thus have a dual influence depending on the considered organ. The inhibitor of ABA synthesis fluridone reduced endogenous ABA but did not compromise ferritin gene expression or ferritin synthesis, whatever the iron dose. Methyviologen application induced obvious oxidative damages but reduced ferritin synthesis. It is suggested that the signalling pathway leading to ferritin synthesis in the semi-aquatic African rice species may involve other components than those reported for typical terrestrial plants.     

Oxidation-induced ferritin turnover in microglial cells: role of proteasome

Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules.     

Effect of actinomycin D, cycloheximide, and acute blood loss on ferritin synthesis in rat liver

1. The mechanism of the stimulation of ferritin synthesis by iron in vivo has been studied in rat liver. Ferritin synthesis and turnover was measured by [(14)C]leucine incorporation. 2. Actinomycin D had no inhibitory effect, after administration of iron, on [(14)C]leucine incorporation into ferritin but appeared to augment the effect of iron on ferritin synthesis. 3. Cycloheximide completely abolished the stimulation by iron of [(14)C]leucine into ferritin and was subsequently utilized to show that iron acts in vivo by translational induction of apoferritin synthesis, rather than by stabilization of apoferritin or its precursors. 4. This conclusion was confirmed by showing that 2 days after acute bleeding, when iron was in the process of being removed from hepatic ferritin stores, ferritin synthesis was decreased whereas breakdown rates were unchanged.     

A human mitochondrial ferritin encoded by an intronless gene

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.     

The many faces of the octahedral ferritin protein

Iron is an essential trace nutrient required for the active sites of many enzymes, electron transfer and oxygen transport proteins. In contrast, to its important biological roles, iron is a catalyst for reactive oxygen species (ROS). Organisms must acquire iron but must protect against oxidative damage. Biology has evolved siderophores, hormones, membrane transporters, and iron transport and storage proteins to acquire sufficient iron but maintain iron levels at safe concentrations that prevent iron from catalyzing the formation of ROS. Ferritin is an important hub for iron metabolism because it sequesters iron during times of iron excess and releases iron during iron paucity. Ferritin is expressed in response to oxidative stress and is secreted into the extracellular matrix and into the serum. The iron sequestering ability of ferritin is believed to be the source of the anti-oxidant properties of ferritin. In fact, ferritin has been used as a biomarker for disease because it is synthesized in response to oxidative damage and inflammation. The function of serum ferritin is poorly understood, however serum ferritin concentrations seem to correlate with total iron stores. Under certain conditions, ferritin is also associated with pro-oxidant activity. The source of this switch from anti-oxidant to pro-oxidant has not been established but may be associated with unregulated iron release from ferritin. Recent reports demonstrate that ferritin is involved in other aspects of biology such as cell activation, development, immunity and angiogenesis. This review examines ferritin expression and secretion in correlation with anti-oxidant activity and with respect to these new functions. In addition, conditions that lead to pro-oxidant conditions are considered.     

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.     

The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site

Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry. The only known exception is the dodecameric ferritin from Listeria innocua. The structure of Listeria ferritin has been determined to a resolution of 2.35 A by molecular replacement, using as a search model the structure of Dps from Escherichia coli. The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes. Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites. The L. innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.