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.     

A Quantitative Analysis of Isoferritins in Select Regions of Aged, Parkinsonian, and Alzheimer's Diseased Brains

Abstract: The brain requires a ready supply of iron for normal neurological function, but free iron is toxic. Consequently, iron bioavailability must be stringently regulated. Recent evidence has suggested that the brain iron regulatory system is dysfunctional in neurological disorders such as Alzheimer's and Parkinson's diseases (AD and PD, respectively). A key component of the iron regulatory system in the brain is ferritin. Ferritin consists of 24 subunits, which are distinguished as either a heavy-chain (H) or light-chain (L) isoform. These peptide subunits are genetically and functionally distinct. Thus, the ability to investigate separately the types of ferritin in brain should provide insight into iron management at both the cellular and the molecular level. In this study, the ratio of isoferritins was determined in select regions of adult elderly AD and PD human brains. The H-rich ferritin was more abundant in the young brain, except in the globus pallidus where the ratio of H/L ferritin was 1:1. The balance of H/L isoferritins was influenced by age, brain region, and disease state. With normal aging, both H and L ferritin increased; however, the age-associated increase in isoferritins generally failed to occur in AD and PD brain tissue. The imbalance in H/L isoferritins was disease and region specific. For example, in frontal cortex, there was a dramatic (fivefold) increase in the ratio of H/L ferritin in AD brains but not in PD brains. In PD, caudate and putamen H/L ratios were higher than in AD and the elderly control group. The analysis of isoferritin expression in brain provides insight into regional iron regulation under normal conditions and suggests a loss of ability to maintain iron homeostasis in the two disease states. This latter observation provides further evidence of dysfunction of iron homeostatic mechanisms in AD and PD and may contribute significantly to understanding the underlying pathogenesis of each, particularly in relation to iron-induced oxidative damage.     

Subunit dimers in sheep spleen apoferritin. The effect on iron storage

Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the protein shell in determining the iron storage properties of ferritin.     

Early embryonic lethality of H ferritin gene deletion in mice

Ferritin molecules play an important role in the control of intracellular iron distribution and in the constitution of long term iron stores. In vitro studies on recombinant ferritin subunits have shown that the ferroxidase activity associated with the H subunit is necessary for iron uptake by the ferritin molecule, whereas the L subunit facilitates iron core formation inside the protein shell. However, plant and bacterial ferritins have only a single type of subunit which probably fulfills both functions. To assess the biological significance of the ferroxidase activity associated with the H subunit, we disrupted the H ferritin gene (Fth) in mice by homologous recombination. Fth(+/-) mice are healthy, fertile, and do not differ significantly from their control littermates. However, Fth(-/-) embryos die between 3.5 and 9.5 days of development, suggesting that there is no functional redundancy between the two ferritin subunits and that, in the absence of H subunits, L ferritin homopolymers are not able to maintain iron in a bioavailable and nontoxic form. The pattern of expression of the wild type Fth gene in 9.5-day embryos is suggestive of an important function of the H ferritin gene in the heart.     

Ferritin and ferritin isoforms II: protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes

Ferritin is a major iron storage protein involved in the regulation of iron availability. Each ferritin molecule comprises 24 subunits. Various combinations of H-subunits and L-subunits make up the 24-subunit protein structure and these ferritin isoforms differ in their H-subunit to L-subunit ratio, as well as in their metabolic properties. Ferritin is an acute-phase protein and its expression is up-regulated in conditions such as uncontrolled cellular proliferation, in any condition marked by excessive production of toxic oxygen radicals, and by infectious and inflammatory processes. Under such conditions ferritin up-regulation is predominantly stimulated by increased reactive oxygen radical production and by cytokines. The major function of ferritin in these conditions is to reduce the bio-availability of iron in order to stem uncontrolled cellular proliferation and excessive production of reactive oxygen radicals. Ferritin is not, however, indiscriminately up-regulated in these conditions as a marked shift towards a predominance in H-subunit rich ferritins occurs. Preliminary indications are that, while the L-subunit primarily fulfils the conventional iron storage role, the H-subunit functions primarily as rapid regulator of iron availability, and perhaps indirectly as regulator of other cellular processes. It is suggested that the optimum differential expression of the two subunits differ for different cells and under different conditions and that the expression of appropriate isoferritins offers protection against uncontrolled cellular proliferation, oxidative stress and against side effects of infectious and inflammatory conditions.     

The effects of ascorbic acid on the intracellular metabolism of iron and ferritin

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.     

Ferritin: an iron storage protein with diverse functions

Ferritin is the major protein for iron storage and iron detoxification. Since non-ferrous metals, such as aluminum, beryllium and zinc, are bound both in vivo and in vitro, ferritin is implicated as a general metal ion donor and detoxicant. The role of ferritin in Al and Be toxicity is discussed. During iron release ferritin produces free radicals which are involved in phosphoprotein inactivation, lipid peroxidation and, possibly, the general aging process. Conversely, during iron loading oxidative energy in the form of electrons and protons is given off. The different subunit compositions of ferritin, termed isoferritins, are, at least in part, involved with the multifunctionality of this protein.     

Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin

Ferritin is a multisubunit protein, controlling iron storage, with a protein coat composed of 24 subunits (up to three distinct types) in different proportions depending on cell type. Little is known about the subunit interactions in ferritin protein coats composed of heterologous subunits, despite the relevance to ferritin structure and ferritin function (iron uptake and release). Synthetic crosslinking is a convenient way to probe subunit contacts. Crosslinks between subunit pairs in ferritin protein coats are also a natural post-translational modification which coincides with different iron content in ferritin from sheep spleen; ferritin from sheep spleen also contains H and L subunits. Crosslinks synthesized by the reaction of ferritin low in natural crosslinks with difluorodinitrobenzene (F2DNB) reproduced the effects of the natural crosslinks on iron uptake and release. We now extend our observations on the structural effects of natural and synthetic crosslinks to include immunoreactivity of the assembled protein, with monoclonal antibodies as a probe. We also demonstrate, for the first time, ferritin peptides involved in an apparent H- and L-subunit contact: two peptides decreased 4X in cyanogen bromide peptide maps after F2DNB crosslinking were residues L-96-138 and H-66-96; the major DNP-dipeptide was Lys-DNP-Lys. Using the structure of an all L-subunit ferritin as a model, the most likely site for the H-L DNP crosslink is L-Lys 104 (C helix) and H-Lys 67 (B helix). The B helix forms the internal subunit dimer interface, a putative site of iron core nucleation. Alteration by crosslinks of the B helix could, therefore, explain the effect of crosslinks on ferritin iron uptake, release, and iron content.     

Characterization of ferritin in murine erythroleukaemia cells

Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No change was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the 'last-in-first-out' principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions.     

A novel plant ferritin subunit from soybean that is related to a mechanism in iron release

Ferritin is a multimeric iron storage protein composed of 24 subunits. Ferritin purified from dried soybean seed resolves into two peptides of 26.5 and 28 kDa. To date, the 26.5-kDa subunit has been supposed to be generated from the 28-kDa subunit by cleavage of the N-terminal region. We performed amino acid sequence analysis of the 28-kDa subunit and found that it had a different sequence from the 26.5-kDa subunit, thus rendering it novel among known soybean ferritins. We cloned a cDNA encoding this novel subunit from 10-day-old seedlings, each of which contained developed bifoliates, an epicotyl and a terminal bud. The 26.5-kDa subunit was found to be identical to that identified previously lacking the C-terminal 16 residues that correspond to the E helix of mammalian ferritin. However, the corresponding region in the 28-kDa soybean ferritin subunit identified in this study was not susceptible to cleavage. We present evidence that the two different ferritin subunits in soybean dry seeds show differential sensitivity to protease digestions and that the novel, uncleaved 28-kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5-kDa subunit. These data demonstrate that soybean ferritin is composed of at least two different subunits, which have cooperative functional roles in soybean seeds.     

The distribution of ferritin subunit types in porcine tissues

Ferritin was isolated from porcine heart, liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits yielded three chromatographic fractions. The relative proportions of the three chromatographic fractions were different for each tissue ferritin. These results support the model which proposes a combination of (at least) two subunit types as the basis for the existence of isoferritins.     

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.     

Overexpression of H ferritin and up-regulation of iron regulatory protein genes during differentiation of 3T3-L1 pre-adipocytes

The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.     

Translational regulation of ferritin synthesis in rat liver. Effects of chronic dietary iron overload.

In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.