Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Transferrin as a donor of iron to mitochondria. Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

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.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Uptake and release of ferritin iron. Surface effects and exchange within the crystalline core

The uptake and subsequent release of iron by apoferritin and ferritin was studied by using labelled iron ((59)Fe). The experimental results are consistent with predictions arising from a model system developed in the interpretation of previous experiments. In this model, uptake and release of ferritin iron is controlled by the available surface area of the small crystalline particles of hydrous ferric oxide found within the ferritin molecule. Evidence is also presented for the exchange of Fe(3+) ions among the various cation sites within these crystallites.     

Iron uptake mediated by binding of H-ferritin to the TIM-2 receptor in mouse cells

Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with (55)Fe and (55)Fe-HFt was incubated with TIM-2 positive cells or controls. (55)Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of (55)Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.     

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

Biochemistry of nonheme iron in man. I. Iron proteins and cellular iron metabolism

Total plasma iron turnover in man is about 36 mg/day. Transferrin is the iron transport protein of plasma, which can bind 2 atoms of iron per protein molecule, and which interacts with various cell types to provide them with the iron required for their metabolic and proliferative processes. All tissues contain transferrin receptors on their plasma membrane surfaces, which interact preferentially with diferric transferrin. In erythroid cells as well as certain laboratory cell lines, the removal of iron from transferrin apparently proceeds via the receptor-mediated endocytosis process. Transferrin and its receptor are recycled to the cell surface, whereas the iron remains in the cell. The mode of iron uptake in the hepatocyte, the main iron storage tissue, is less certain. The release of iron by hepatocytes, as well as by the reticuloendothelial cells, apparently proceeds nonspecifically. All tissues contain the iron storage protein ferritin, which stores iron in the ferric state, though iron must be in the ferrous state to enter and exit the ferritin molecule. Cellular cytosol also contains a small-molecular-weight ferrous iron pool, which may interact with protoporphyrin to form heme, and which apparently is the form of iron exported by hepatocytes and macrophages. In plasma, the ferrous iron is converted into the ferric form via the action of ceruloplasmin.     

Iron absorption in the iron-deficient rat

Iron absorption in the iron-deficient rat was compared with that in the normal rat to better understand the regulation of this dynamic process. It was found that: Iron uptake by the iron-deficient intestinal mucosa was prolonged as a result of slower gastric release, particularly when larger doses of iron were employed. The increased mucosal uptake of ionized iron was not the result of increased adsorption, but instead appeared related to a metabolically active uptake process, whereas the increased mucosal uptake of transferrin iron was associated with increased numbers of mucosal cell membrane transferrin receptors. Mucosal ferritin acted as an iron storage protein, but its iron uptake did not explain the lower iron absorption in the normal rat. Iron loading the mucosal cell (by presenting a large iron dose to the intestinal lumen) decreased absorption for 3 to 4 days. Iron loading of the mucosal cell from circulating plasma transferrin was proportionate to the plasma iron concentration. Mucosal iron content was the composite of iron loading from the lumen and loading from plasma transferrin versus release of iron into the body. These studies imply that an enhanced uptake-throughout mechanism causes the increased iron absorption in the iron-deficient rat. Results were consistent with the existence of a regulating mechanism for iron absorption that responds to change in mucosal cell iron, which is best reflected by mucosal ferritin.     

Iron uptake mechanism in the chrysophyte microalga Dinobryon

The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.     

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell

Background

Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.

Scope of review

Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.

Conclusions and general significance

These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.     

Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa

Metabolism of iron derived from insoluble and/or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O₂ or H₂O₂. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high.     

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.     

Iron mobilization from cultured rat bone marrow macrophages

The reticuloendothelial system is responsible for removing old and damaged erythrocytes from the circulation, allowing iron to return to bone marrow for hemoglobin synthesis. Cultured bone marrow macrophages were loaded with ⁵⁹Fe-labelled erythroblasts and iron mobilization was studied. After erythroblast digestion, iron taken up by macrophages was found in ferritin as well as in a low-molecular-weight fraction. The analysis of iron mobilization from macrophages shows: (1) the iron was mobilized as ferritin. (2) A higher mobilization was observed when apotransferrin was present in the culture medium. (3) In the presence of apotransferrin in the culture medium, part of the iron was found as transferrin iron. (4) Iron transfer from ferritin to apotransferrin was observed in a cell-free culture medium and this process was temperature independent. The results indicate that after phagocytosis of ⁵⁹Fe-labelled erythroblasts by macrophages, iron is mobilized as ferritin. In the plasma, this iron can be transferred to apotransferrin.     

Iron gathering of opportunistic pathogenic fungi. A mini review

Iron is an essential nutrient for most organisms because it serves as a catalytic cofactor in oxidation-reduction reactions. Iron is rather unavailable because it occurs in its insoluble ferric form in oxides and hydroxides, while in serum of mammalian hosts is highly bound to carrier proteins such as transferrin, so the free iron concentration is extremely low insufficient for microbial growth. Therefore, many organisms have developed different iron-scavenging systems for solubilizing ferric iron and transporting it into cells across the fungal membrane. There are three major mechanisms by which fungi can obtain iron from the host: (a) utilization of a high affinity iron permease to transport iron intracellularly, (b) production and secretion of low molecular weight iron-specific chelators (siderophores), (c) utilization of a hem oxygenase to acquire iron from hemin. Patients with elevated levels of available serum iron treated with iron chelator, deferoxamine to remedy iron overload conditions have an increased susceptibility of invasive zygomycosis. Presumably deferoxamine predisposes patients to Zygomycetes infections by acting as a siderophore]. The frequency of zygomycosis is increasing in recent years and these infections respond very poorly to currently available antifungal agents, so new approaches to develop strategies to prevent and treat zygomycosis are urgently needed. Siderophores and iron-transport proteins have been suggested to function as virulence factors because the acquisition of iron is a crucial pathogenetic event. Biosynthesis and uptake of siderophores represent possible targets for antifungal therapy.     

Iron accumulation and iron-regulatory protein activity in human hepatoma (HepG2) cells

Iron may populate distinct hepatocellular iron pools that differentially regulate expression of proteins such as ferritin and transferrin receptor (TfR) through iron-regulatory mRNA-binding proteins (IRPs), and may additionally regulate uptake and accumulation of non-transferrin-bound iron (NTBI). We examined iron-regulatory protein (IRP) binding activity and ferritin/TfR expression in human hepatoma (HepG2) cells exposed to iron at different levels for different periods. Several iron-dependent RNA-binding activities were identified, but only IRP increased with beta-mercaptoethanol. With exposures between 0 and 20 microg/ml iron, decreases in IRP binding accompanied large changes in TfR and ferritin expression, while chelation of residual iron with deferoxamine (DFO) caused a large increase in IRP binding with little additional effect on TfR or ferritin expression. Cellular iron content increased beyond 4 days of exposure to iron at 20 microg/ml, when IRP binding, TfR, and ferritin had all reached stable levels. However, iron content of the cells plateaued by 7 days, or decreased with 24 h exposure to very high concentrations (>50 microg/ml) of iron. These results indicate that iron-replete HepG2 cells exhibit a narrow range of maximal responsiveness of the IRP-regulatory mechanism, whose functional response is blunted both by excessive iron exposure and by removal of iron from a chelatable pool. HepG2 cells are able to limit iron accumulation upon higher or prolonged exposure to NTBI, apparently independent of the IRP mechanism.     

Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.