InteractiveFly: GeneBrief

Ferritin 1 heavy chain homologue and Ferritin 2 light chain homologue: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Ferritin is a symmetric, 24-subunit iron-storage complex assembled of H and L chains. H and L heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. Ferritin is found in bacteria, plants and animals, and two classes of mutations in the human L-chain gene result in hereditary hyperferritinemia cataract syndrome or in neuroferritinopathy. This study examined systemic and cellular ferritin regulation and trafficking in Drosophila; ferritin H and L transcripts are co-expressed during embryogenesis and both subunits are essential for embryonic development. Ferritin overexpression impairs the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. This study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization (Missirlis, 2007).

Iron is an essential element of aerobic life. Cells have evolved highly regulated molecular pathways that ensure iron incorporation into heme, or formation of iron-sulfur clusters. Cellular and systemic iron levels are tightly regulated to ensure bioavailability and protect from the hazards of iron overload. Ferritin, a heteropolymer composed of H and L subunits, acts as the primary iron storage molecule. The H subunit contains a ferroxidase center, which enables the mature heteropolymer to oxidize soluble ferrous iron, whereas the L chain provides the nucleation centers for deposition of the ferrihydrite mineral (Missirlis, 2007 and references therein).

In mammals, transcriptional control of the ferritin genes influences the relative ratio of H to L chains in different cell types (Torti, 2002; Pham, 2004). Translation of ferritin proteins is regulated by the binding of either of the two iron regulatory proteins (IRPs) to an iron responsive element (IRE) located on the 5’ untranslated region (UTR) of the respective mRNAs (Pantopoulos, 2004; Rouault 2006). Hereditary hyperferritinemia cataract syndrome, a disease in which ferritin L-chain IRE mutations interfere with appropriate translational repression, illustrates the physiological importance of the IRP/IRE system (Cazzola, 2002; Rouault 2006). Moreover, adult mice lacking IRP2 overexpress ferritin, develop variable degrees of late-onset neurodegeneration (Lavaute, 2001; Smith, 2004; Galy, 2006) and anemia (Cooperman, 2005; Galy, 2005). Negative consequences of chronic ferritin H-chain overexpression have been verified in aging mice (Kaur, 2006). Conversely, protection from oxidative stress has been shown in young mice that overexpress ferritin H-chain, because of the iron-chelating properties of ferritin (Kaur, 2003; Wilkinson, 2006). A complete null for ferritin H-chain has been generated in mice; homozygous animals die in utero, whereas heterozygotes exhibit signs of mild iron deficiency (Thompson, 2003). Finally, mutations in the human ferritin L-chain lead to neurodegeneration in a condition described as neuroferritinopathy (Levi, 2005). Altogether, these results underscore the importance of ferritin regulation in mammalian health (Missirlis, 2007).

In Drosophila, Ferritin 1 heavy chain homolog (Fer1HCH) and Ferritin 2 light chain homolog (Fer2LCH) encode the ferritin subunits that compose the major, secreted form of ferritin (Charlesworth, 1997; Georgieva, 1999; Georgieva, 2002). The crystal structure of secreted ferritin from Trichoplusia ni revealed a symmetrical arrangement of H and L chains (Hamburger, 2005). Inter and intrasubunit disulfide bonds were shown to be important for the folding/assembly of Trichoplusia ni ferritin, and the respective cysteine residues mediating these bonds were also conserved in Drosophila melanogaster, suggesting that the ferritins of the two species share the same mode of assembly (Hamburger, 2005). The Fer1HCH amino acid residues that are required for ferroxidase activity in mammals were conserved in the insect ferritin structure, and a predicted Fer2LCH ferrihydrite nucleation site formed by the L-chains was also found (Hamburger, 2005; Missirlis, 2007 and references therein).

Intracellular localization of ferritin in many insects also differs from mammals. Ultra-structural studies, combining electron microscopy and energy electron loss spectroscopy, have revealed the presence of Calpodes ferritin in intracellular membrane compartments (Locke, 1984). Drosophila Fer1HCH and Fer2LCH subunits contain signal peptides that direct them to the endoplasmic reticulum upon translation. Fer1HCH also contains a predicted N-Glycosylation site. The two subunits are predominantly expressed in the midgut and are also abundant in hemolymph, where ferritin may transport iron for nutritional needs of Drosophila tissues (Georgieva; 2002; Missirlis, 2007 and references therein).

This study shows that mutational inactivation of either Fer1HCH or Fer2LCH in Drosophila, as well as the disruption of the ferroxidase center of Fer1HCH, results in developmental arrest and fly embryonic lethality. A novel fly strain expressing GFP-tagged Fer1HCH has been characterized: it was shown that GFP-Fer1HCH is incorporated into endogenous functional ferritin. This strain was used to study induction and trafficking of ferritin in the fly midgut, the major iron-storing organ in the insect (Missirlis, 2007).

Several novel findings on Drosophila ferritin are described in this work. Absence of either Fer1HCH or Fer2LCH results in embryonic lethality and modified Fer1HCH subunits (mutant in the ferroxidase center or GFP-tagged) cannot substitute for lack of Fer1HCH. However, if the same modified subunits are expressed in the presence of wild type subunits, they can be integrated into ferritin holomers without inducing dominant negative effects. Analysis of heterozygous loss-of-function ferritin fly mutants or flies overexpressing ferritin subunits revealed that a constant ratio of Fer1HCH and Fer2LCH is maintained, independent of their internal transcriptional expression levels. The structural cooperation of the two subunits that is secured via disulfide bonds (Hamburger, 2005) likely explains these observations. A posttranscriptional mechanism, possibly involving the degradation of subunits that are present in excess, ensures the presence of equal amounts of the two subunits. Such a mechanism can explain the absence of the IRE in Fer2LCH mRNAs in insects, since Fer1HCH translational repression by IRP-1A under iron-limiting conditions (that favor the IRE-containing transcripts) would then be sufficient to reduce levels of Fer2LCH. In contrast to results from mammalian cell or animal models (Thompson, 2003; Wilkinson, 2006), but consistent with what is known from human patients with hereditary hyperferritinemia cataract syndrome (Cazzola, 2002), experimental reduction or increase of ferritin levels through genetic manipulation in Drosophila caused only very mild alterations in the insect’s iron homeostasis. These results point towards an independent regulatory system that controls iron sequestration into ferritin. The nature of this system is currently unknown, but could involve a putative iron chaperone that delivers iron to ferritin. However, the hypothesized chaperone’s function does not completely override the need for ferritin regulation, as shown by the phenotype of ferritin overexpressing flies that was lethal under low iron conditions but was rescued with iron supplementation. Alternatively, localization of ferritin in the Golgi apparatus of insect cells may prevent it from contact with the cytosolic and mitochondrial iron pools. It is currently not known how iron is delivered to the ferritin that resides in the secretory pathway of cells (Missirlis, 2007).

Evidence is provided that the ferritin genes are co-expressed during embryogenesis. It was wondered whether their genetic proximity is conserved in other Drosophila species with fully sequenced genomes. To this end, EvoPrinter, a new multigenomic DNA sequence analysis tool that facilitates the rapid identification of evolutionarily conserved sequences within the context of a single species, was used (Odenwald, 2005). In silico analysis of the Fer1HCH and Fer2LCH genomic locus by EvoPrinter produced an output of the combined mutational histories of six Drosophila species, superimposed on a reference sequence from Drosophila melanogaster. The results not only showed that clustering of the two genes is conserved, but also identified potentially shared regulatory regions. From the 17670 base pairs spanning the two genes that were analyzed, only 914 (or 5.2%) were conserved in an identical position in all six species tested. One third of these conserved sequences (306) were contained within the open reading frames. Surprisingly, over half of them (491) were not contained in the cDNA sequence, but were rather clustered in two regions: downstream of the Fer2LCH transcribed region or in the second intron of Fer1HCH. A particular stretch of ten base pairs, TTTGCACACG, was found three times in the second intron of Fer1HCH and could represent a binding site for an unidentified factor. The remaining conserved base pairs (117) were mostly concentrated in the 5’ UTR of Fer1HCH, including the IRE itself. The conservation of the IRE over approximately 160 million years of collective evolutionary divergence underscores the functional significance of the IRE/IRP control system in all Drosophila species (Missirlis, 2007).

Ferritin regulation is a critical aspect of the organism’s iron economy. The finding that ferritin expression in the iron region is constitutive (and remains so even under iron deficient conditions), but is inducible in the anterior midgut has a parallel with the expression of metallothioneins, which are the copper storage proteins. Interestingly, cuprophilic cells that are present anterior to the iron region show constitutive metallothionein expression, whereas cells in both the anterior and posterior midgut induce metallothionein expression and copper/metallothionein fluorescence at higher copper concentrations. Results presented in this paper combined with previous reports, lead to the following fundamental conclusion with respect to metal metabolism in the midgut of Drosophila: the insect midgut contains two sets of specialized cells, one of which constitutively expresses metallothionein and a second constitutively expresses ferritin. If either metal is present in great abundance, a third and fourth set of distinct midgut cells have the potential to induce transcription of the genes that encode the two metal storage proteins. Importantly, there are also cells in the midgut that do not respond significantly to high concentrations of these metals. Whether similar cellular populations exist in the mammalian intestine remains unknown, but cellular populations with specific metal contents have been described in plant seeds (Missirlis, 2007).

GFP-tagged ferritin revealed the complex physiologic orchestration of intestinal metal responses. In addition, it has enabled visualization of subcellular ferritin dynamics upon iron-mediated induction and should facilitate the dissection and subcellular localization of ferritin biosynthesis and trafficking. Serum ferritin is measured in clinical practice as a measure of total body iron stores (Beutler, 2002) and is an acute phase reactant (Tran, 1997) but few studies have addressed how ferritin is secreted in the circulatory system of humans (Ghosh, 2004; Renaud, 1991). Importantly, ferritin tagged with GFP could function and elucidate trafficking mechanisms in human cells and transgenic mice (Missirlis, 2007).

Ferritin overexpression in Drosophila glia leads to iron deposition in the optic lobes and late-onset behavioral defects

Cellular and organismal iron storage depends on the function of the ferritin protein complex in insects and mammals alike. In the central nervous system of insects, the distribution and relevance of ferritin remain unclear, though ferritin has been implicated in Drosophila models of Alzheimers' and Parkinsons' disease and in Aluminum-induced neurodegeneration. This study shows that transgene-derived expression of ferritin subunits in glial cells of Drosophila melanogaster causes a late-onset behavioral decline, characterized by loss of circadian rhythms in constant darkness and impairment of elicited locomotor responses. Anatomical analysis of the affected brains revealed crystalline inclusions of iron-loaded ferritin in a subpopulation of glial cells but not significant neurodegeneration. Although transgene-induced glial ferritin expression was well tolerated throughout development and in young flies, it turned disadvantageous at older age. The flies characterized in this report contribute to the study of ferritin in the Drosophila brain and can be used to assess the contribution of glial iron metabolism in neurodegenerative models of disease (Kosmidis, 2011).

The accumulation of iron-loaded ferritin in glia when both ferritin subunits were overexpressed simultaneously suggests that mis-regulation of brain iron homeostasis may result in failure to maintain circadian rhythms and an overall behavioural decline at late age. The anatomical location of the ferritin inclusions raises the intriguing possibility that the function of the Hofbauer-Buchner eyelet, previously shown to participate in the synchronization and entrainment of natural rhythms, might be affected as a consequence of ferritin aggregation. Indeed, the eyelet sends projections through the chiasma between the lamina and medulla and might be physically or metabolically constrained by the presence of crystalline formations of iron-loaded ferritin in surrounding glia. Experiments in this study support a previously suggested role of glia in the maintenance of the circadian clock. It isd proposed that Drosophila is a useful model to elucidate interactions between iron homeostasis and the endogenous clock (Kosmidis, 2011).

A poorly understood connection between brain iron metabolism and the dopaminergic neural network is sometimes invoked in explanations of the Restless legs syndrome (RLS) pathology. RLS is a condition characterized by circadian timing of movement disorder. Some patients with RLS respond well to dietary iron supplementation. Tyrosine hydroxylase, an iron-dependent enzyme in the dopamine biosynthetic pathway, may be induced despite (or as a consequence of) systemic iron deficiency, a finding that remains poorly understood. As iron deficiency is a serious risk factor for developing RLS, it will be interesting to investigate whether the iron accumulation in glial ferritin inclusions, upon ferritin overexpression, may deplete bioavailable iron for other cellular functions. Future experiments aimed to uncover links between ferritin, the circadian clock and neuroaminergic outputs in the fly brain will hopefully enhance understanding of the role of iron in the numerous human neurological disorders it has been already implicated (Kosmidis, 2011).

REGULATION

Ferritin Assembly in Enterocytes of Drosophila melanogaster

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. This study addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. Concentration was placed on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. The expression pattern of Fer2LCH-Gal4 lines were shown to recapitulate iron-dependent endogenous expression of the ferritin subunits; and these lines were used to drive expression from UAS-mCherry-Fer2LCH transgenes. The Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. This study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated (Rosas-Arellano, 2016).

Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis

Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multiple oxidases, MCO1-4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. This study shows Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. The likely existence of a similar iron efflux apparatus is speculated as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway (Wang, 2018).

Transcriptional Regulation

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion were determined. Furthermore, the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant was analyzed. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in this study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification (Yepiskoposyan, 2006).

An unexpected result of the present study is the induction of ferritin heavy and light chain homologue genes by heavy metals. Ferritin is well known to detoxify, store and transport iron. It was also shown to bind other metal ions and has been speculated to function as a general metal detoxicant in mammals. The results suggest an induction of Drosophila ferritins by metals other than iron, notably zinc. The latter induction depends on MTF-1, based on the following findings: (1) the presence of multiple MREs, (2) the loss of zinc induction in the MTF-1 mutant, and (3) elevated ferritin transcripts upon MTF-1 overexpression. In contrast, iron inducibility must be regulated independently since it is not altered by the absence of MTF-1. In line with different signaling pathways responding to iron load versus excess of zinc, copper or cadmium, it is noted that the classical MTF-1 dependent MtnA gene is not inducible by iron. Yet these pathways apparently overlap in the ferritin genes (Yepiskoposyan, 2006).

Iron response elements in Drosophila

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. IREs have been identified and characterized in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. These results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis (Gray, 1996; Missirlis, 2007).

As in vertebrates, the IRE/IRP system functions in Drosophila (Rothenberger, 1990; Missirlis, 2003). A functional IRE is present in the 5’UTR of the Fer1HCH mRNA, but only in certain splice variants that are preferentially encoded under iron-limiting conditions (Lind, 1998; Georgieva, 1999). In contrast, no IRE is present in Fer2LCH mRNA (Georgieva, 2002). IRP homologs are expressed in the fly (Muckenthaler, 1998) and one homolog (IRP-1A) has been shown to bind to IREs from both Drosophila and mammals (Lind, 2006; Missirlis, 2007).

Drosophila IRPs

Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans, have been identifed. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network (Muckenthaler, 1998).

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, a genetic analysis of the interaction between iron and oxygen metabolism was undertaken in Drosophila. Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. Exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, it was found that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. It is concluded that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments (Missirlis, 2003).

In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs. The Drosophila genome offers an evolutionary intermediate containing two IRP1-like proteins (IRP-1A and IRP-1B) and target genes with IREs. This study used purified recombinant IRP-1A and IRP-1B from Drosophila melanogaster and showed that only IRP-1A can bind to IREs, although both proteins possess aconitase activity. These results were also corroborated in whole-fly homogenates from transgenic flies that overexpress IRP-1A and IRP-1B in their fat bodies. Ubiquitous and muscle-specific overexpression of IRP-1A, but not of IRP-1B, resulted in pre-adult lethality, underscoring the importance of the biochemical difference between the two proteins. Domain-swap experiments showed that multiple amino acid substitutions scattered throughout the IRP1 domains are synergistically required for conferring IRE binding activity. These data suggest that as a first step during the evolution of the IRP/IRE system, the ancient cytosolic aconitase was duplicated in insects with one variant acquiring IRE-specific binding (Lind, 2006).

Ferritin Activity

A GFP-containing P-element integrated into the second intron of Fer1HCH was used to study ferritin expression. Intron/exon donor and acceptor splice sites flank the GFP sequence on the P-element; consequently all Fer2LCH^G188 mRNA types (IRE +/-) are predicted to contain the GFP exon, which should encode GFP in frame with the Fer1HCH protein. Therefore, post-transcriptional regulation of GFP-Fer1HCH mediated by the IRP-1A (Lind, 2006) would remain unaffected by the GFP exon. GFP is inserted two amino acids downstream of the predicted cleavage site for the Fer1HCH signal peptide (Charlesworth, 1997), which is encoded from the second exon of the gene. Thus, the full-length mature polypeptide is predicted to translocate to the endoplasmic reticulum and contain the GFP attached to the N-terminus, after the signal peptide is cleaved. To test whether the GFP-Fer1HCH fusion polypeptide was generated as predicted from the locus containing the G188 P-element, extracts from adult wild type (+/+) flies or flies heterozygous for the GFP insertion line (G188/+) were separated by SDS-page electrophoresis under reducing conditions. Western blots were probed with antisera raised against Fer1HCH or Fer2LCH peptides. Results indicated the presence of two immuno-reactive bands with the Fer1HCH antibody in Fer2LCH^G188/+ lysates. The higher-molecular weight species migrated at the predicted size of 50kDa, consistent with a 27kDa GFP addition to the 23kDa Fer1HCH chain. Levels of the Fer2LCH did not change compared to wild type lysates (Missirlis, 2007).

To address whether the expressed GFP-Fer1HCH polypeptide contributes to the formation of the holoferritin in the transgenic insect, the same extracts were separated by SDS-page electrophoresis under non-reducing conditions, which have previously been shown to preserve higher molecular weight ferritin heteropolymers (Missirlis, 2006). In extracts from wild type flies a single ferritin species was observed that migrated close to mouse liver ferritin. As expected from the lower levels of both ferritin subunits in Fer1HCH^451/+ heterozygotes, these flies expressed less total ferritin. Conversely, in extracts from heterozygous Fer2LCH^G188/+ flies that expressed GFP-tagged ferritin, several higher-molecular weight bands could be detected. Thus, ferritin polymers are formed in these flies and the different molecular weight species most likely reflect varying ratios of Fer1HCH and GFP-Fer1HCH chains in polymers. Importantly, no ferritin composed solely of native Fer1HCH and Fer2LCH was detected in the heterozygous Fer2LCH^G188/+ flies, suggesting that the GFP-tagged ferritins are the functional ferritins of these animals. However, homozygous Fer2LCH^G188/G188 and hetero-allelic Fer2LCH^G188/451 embryos failed to develop into larvae, indicating that the fly cannot survive when all Fer1HCH subunits are tagged with GFP. It was of interest to find out if GFP tagging of all Fer1HCH subunits hindered ferritin assembly, or whether it obstructed access of an iron carrier to the assembled ferritin. If failure to assemble ferritin with 12 GFP-Fer1HCH subunits was the cause of lethality, rescue or the lethality of Fer2LCH^G188/451 flies by overexpression of the mutant ferroxidase construct UAS-Fer1HCH* was expected. This expectation arose from the fact that GFP-Fer1HCH should provide intact ferroxidase centers to the holomer, and mutant Fer1HCH* should allow normal assembly. However, overexpression of wild type UAS-Fer1HCH rescued the lethality of Fer2LCH^G188/451 flies, but UAS-Fer1HCH* did not, suggesting that a more likely explanation for the lethality of embryos expressing only GFP-Fer1HCH subunits is that the GFP tag blocks Fer1HCH-interacting proteins from accessing the ferroxidase centers of holoferritin (Missirlis, 2007).

Attempts were made to demonstrate that the GFP-tagged ferritin in the Fer2LCH^G188/+ heterozygous flies is indeed able to sequester iron and substitute for non-tagged holoferritin. To this end, adult flies were fed with radioactive iron in the form of 55FeCl3. Nitrilotriacetate was added to maintain 55Fe3+ in solution at the neutral pH of food, and whole-fly homogenates were prepared after the insects were allowed to feed for 24 hours. Autoradiographs of the non-denaturing SDS-page gels clearly indicated that iron is incorporated in ferritin from wild type or Fer1HCH^451/+ flies but also in GFP-tagged ferritin. The observation that the same amount of iron is associated with half the amount of ferritin protein in the heterozygous Fer1HCH^451/+ strain suggests that the iron load per ferritin holomer can increase when total ferritin decreases (Missirlis, 2007).

Also, the absence of 55Fe incorporation in the heterozygous Fer2LCH^G188/+ strain at the same molecular weight where putative residual normal ferritins would run suggests that these flies survive on ferritin containing both GFP-tagged and non-tagged Fer1HCH subunits. It is speculated that GFP may partially inhibit iron loading is consistent with findings that assembled ferritin molecules with fewer GFP-tags (distinguished by their faster migration) are more heavily iron loaded than assembled ferritin molecules with many GFP-Fer1HCH subunits (Missirlis, 2007).

Thus, although the GFP-Fer1HCH in solo is not fully functional, it coassembles in vivo with Fer1HCH to form functional fluorescent ferritin (Missirlis, 2007).

Overexpression of ferritin requires co-expression of both H and L subunits

Since iron overload is known to induce ferritin expression in vertebrates and flies alike, attempts were made to test the consequences of overexpression of either ferritin H or L genes under normal or low iron levels. Immunoblotting of lysates prepared from whole fly extracts revealed that ubiquitous overexpression of either of the two single chains individually is not sufficient to significantly alter total ferritin amounts or the relative ratio between the two chains. In contrast, when both UAS-Fer1HCH and UAS-Fer2LCH are simultaneously activated with the same Actin-Gal4 driver, robust overexpression is achieved in both sexes. Expression of the ferroxidase inactive UAS-Fer1HCH* was also demonstrated when co-expressed with UAS-Fer2LCH. Overexpression of ferritin was also revealed when the proteins were separated in nonreducing gels. Successful expression from the transgenes was confirmed by performing RT-PCR using primers that were specific to the transgenic mRNA (Missirlis, 2007).

Phenotypes associated with ferritin overexpression: Ferritin overexpression in mammalian cells causes functional iron deficiency due to iron chelation (Cozzi, 2000; Wilkinson, 2006). However, no significant alteration of total radioactive iron associated with overexpressed ferritin was detected after 24-hours of feeding. If the feeding period was extended for 5 days, a slight increase was seen in sequestration of radiolabeled iron into the ferritin of overexpressors, but overall the results show that, at least in Drosophila, ferritin protein levels are not the determining factor controlling iron storage into ferritin. Overexpression of ferroxidase-inactive UAS-Fer1HCH* together with UAS-Fer2LCH in the presence of functional endogenous Fer1HCH subunits results in ferritin heteropolymers that are still potent iron storage complexes (Missirlis, 2007).

It has been shown that overexpression of Fer3HCH, a homopolymeric mitochondrial ferritin composed entirely of Fer3HCH chains, causes female-specific resistance to paraquat (Missirlis, 2006). This study shows that overexpression of Fer1HCH or Fer2LCH alone is not sufficient to confer paraquat resistance, but coexpression of either Fer1HCH or Fer1HCH* in concert with Fer2LCH confers greater survival to a paraquat challenge, underscoring the functional cooperation of the ferritin subunits in vivo. As with flies overexpressing mitochondrial ferritin, resistance to oxidative stress is not observed in males that overexpress Fer1HCH and Fer2LCH. These results were corroborated by using different driver lines (FB-Gal4 and Elav-Gal4) and also by using hydrogen peroxide as a stressor. It is speculated that overexpression of ferritin triggers a developmental signal, possibly related to iron availability, that is specific in females and could relate to the complex nutritional regulation that allocates resources towards reproduction, energy storage or metabolic activity, all adaptive traits in females. In contrast to Fer3HCH overexpression, which does not affect development, flies overexpressing Fer1HCH and Fer2LCH are at a developmental disadvantage compared to their siblings. The progeny from the cross Actin-Gal4/Cyo x UASFer1HCH, UAS-Fer2LCH/UAS-Fer1HCH, UAS-Fer2LCH were scored by gender and by presence or absence of the Actin-Gal4 driver. Two independent sets of recombinant chromosomes were used (one on the X and one on the 3rd) for UAS-Fer1HCH and UAS-Fer2LCH; also flies were used that carried both recombinant chromosomes, allowing for testing if the phenotype was dosage-sensitive. In male flies, the competitive disadvantage of ferritin overexpression is more pronounced than in females. Importantly, the effects of ferritin overexpression are more dramatic under iron-limiting conditions induced by addition of the iron chelator Bathophenanthroline disulfate in the food, whereas the lethal effects can be rescued by dietary iron supplementation. Therefore, the results indicate that ubiquitous ferritin overexpression in the absence of iron overload can be deleterious, by a mechanism which implicates the iron-chelating properties of ferritin (Missirlis, 2007).

DEVELOPMENTAL BIOLOGY

Insect ferritins have subunits homologous to the heavy and light chains of vertebrate ferritins. Cloning and sequence of the heavy chain homologue (HCH) of Drosophila ferritin subunit has been reported earlier. When Northern blots of D. melanogaster RNA were probed with a cDNA for this HCH, three bands were observed. It was shown that these represented at least four classes of mRNA of various lengths. The polymorphism results from alternative splicing of an intron in the 5' untranslated region (UTR) that contains the iron-responsive element (IRE) and from two alternative polyadenylation sites in the 3' UTR. By hybridizing Northern blots with specific probes, it has been shown that the relative proportions of the messages vary with the life stage and especially with iron supplementation of the diet. Iron significantly increases the amount of ferritin HCH messages and dramatically shifts the balance toward those messages that lack an IRE and/or have a short 3' UTR. In the larvae this change takes place in the gut , but not in the fat body. It is speculated that this dramatic shift in message distribution may result from an effect of iron on the rate of transcription or message degradation, or from an effect on the splicing process itself. Synthesis of ferritin HCH subunit mRNAs that lack an IRE may be important under conditions of iron overload (Georgieva, 1999).

Drosophila melanogaster secreted ferritin, like the cytosolic ferritins of other organisms, is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). The cloning of a cDNA encoding the ferritin LCH is repored. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues (Georgieva, 2002).

The genomic proximity between Fer1HCH and Fer2LCH could facilitate similar expression patterns to coordinate biosynthesis of the ferritin heteropolymer. In situ hybridizations of anti-sense RNA probes against the two genes in whole mount preparations of embryos at different stages of development were performed. It was reasoned that if the predominant sites of Fer1HCH and Fer2LCH expression were different, it would be unlikely that they shared common regulatory enhancers, as previously suggested (Dunkov, 1999; Dunkov, 2006; Missirlis, 2007).

Nevertheless, it was found that the different transcripts are expressed in highly specific, yet similar, patterns during embryogenesis. Fer1HCH and Fer2LCH were expressed during oogenesis and the maternal transcripts became evenly distributed in the egg and early embryo, where they were detected in the blastoderm. Tissue-specific transcripts were first detected during germ band elongation in cells of the mesoderm, which are specified to give rise to the fat bodies and amnioserosa. Staining was also seen in cells destined to become macrophages in the anterior head region of embryos. During germ-band retraction and dorsal closure, the amnioserosa retained ferritin mRNAs. At late stages of embryogenesis, cells in the developing midgut initiate ferritin transcription. The similar embryonic expression patterns of Fer1HCH and Fer2LCH suggest that both ferritin subunits are expressed in each cell type where ferritin is required (Missirlis, 2007).

Ferritin is abundant in the midguts from several different insect species and its expression is induced by dietary iron (Capurro Mde, 1996; Dunkov, 2002; Georgieva, 2002; Kim, 2002). It has been shown that GFP-tagged ferritin stores iron in Fer2LCH^G188/+ flies. The expression pattern of GFPtagged ferritin was determined to see if is similar to that of the endogenous non-tagged protein. Indeed, GFPtagged ferritin is most prominently expressed in a cluster of cells of the middle midgut. These cells have been identified as the iron region of the insect midgut on the basis of their positive stain with Prussian blue and the accumulation of exogenously administered radioactive iron. Mid-third instar larvae were administered a diet containing 5mM ferric ammonium citrate and their intestines were dissected for imaging. GFP-tagged ferritin was inducible only in cells of the anterior midgut, but was constitutively expressed in the iron-region of the middle midgut and no expression was detected in the copper cells that are present in-between the two regions (Missirlis, 2007).

Midguts from larvae subjected to the same treatment were also stained with Prussian blue. A light blue staining indicative of the presence of ferric iron was observed in the iron region of both iron-fed and control larvae and was largely unchanged by feeding on iron-enriched diet. In contrast, the anterior region was not stained in control larvae, but stained dark blue in iron-fed individuals, indicating that the ferritin that accumulates in the anterior midgut upon iron feeding is rich in iron content (Missirlis, 2007).

To assess the time frame of ferritin induction during iron feeding and to further demonstrate that a specific subset of cells in the anterior midgut shows a strong response to iron levels, endogenous ferritin levels were determined in a time course following iron feeding. For this experiment wild type third instar larvae (100 hours old at 25°C) were used, and their anterior and middle midguts were dissected for Western Blot analysis. The results showed a clear induction of the ferritin heteropolymers three hours post-feeding in the anterior midgut. Consistent with imaging and iron-staining results, the induction of ferritin was largely restricted to the anterior midgut and was much less pronounced in the iron region (Missirlis, 2007).

Another essential transition metal and dietary nutrient for Drosophila is copper. Copper-containing (cuprophilic) cells function in the acidification of the midgut and are present at different sites than the iron region. Coppermetallothionein complexes in these cells fluoresce orange-red upon ultraviolet illumination. The coppermetallothionein fluorescence and GFP-Ferritin were imaged simultaneously in midguts from larvae fed a diet containing 1mM Cu2+. The results showed that ferritin-expressing cells indeed form a distinct cellular population from cells that contain copper (Missirlis, 2007).

To investigate which cellular compartment accumulates iron-loaded ferritin in midgut cells, GFP-Fer1HCH expressing cells were co-stained with an antibody raised against the Golgi-associated protein Lava lamp. Confocal images from a single cell in the iron region clearly showed that all GFP-ferritin localized within the Golgi compartment, consistent with the images obtained by electron microscopy (Locke, 1984; Nichol, 1990). A few Golgi bodies in cells from the iron region were devoid of ferritin. The same cells were also imaged stained with antibodies against the Fer2LCH subunits. As expected from their tight association revealed by structural studies, biochemical analysis and electron microscopy, there was complete co-localization with GFP-Fer1HCH in the Golgi complex of these specialized cells. Finally, focus was placed on the cells of the anterior midgut that do not normally express ferritin, but potently do so in the presence of high iron levels. The cells were stained for both ferritin chains during the time course of ferritin induction. Prior to iron feeding, GFP-Fer1HCH was not expressed and low levels of Fer2LCH were detected. At one hour post-induction GFP-Fer1HCH was detected in a compartment resembling the endoplasmic reticulum, but Fer2LCH-positive Golgi that were devoid of GFP-Fer1HCH subunits could not be detected. In contrast, four hours post-induction the two subunits were strongly induced and were seen only in complex with one another within the Golgi. Collectively, these results have identified different specialized intestinal sites for iron and copper metabolism and shown that ferritin synthesis is differentially regulated along the intestine. Significantly, the results also validate the use of the Fer2LCH^G188 line as a faithful reporter of endogenous ferritin expression (Missirlis, 2007).

Iron homeostasis controls myeloid blood cell differentiation in Drosophila

Iron is an essential divalent ion for aerobic life. Life has evolved to maintain iron homeostasis for normal cellular and physiological functions and therefore imbalances in iron levels exert a wide range of consequences. Responses to iron dysregulation in blood development, however, remain elusive. This study found that iron homeostasis is critical for differentiation of Drosophila blood cells in the larval hematopoietic organ, called the lymph gland. Supplementation of an iron chelator, bathophenanthroline disulfate (BPS) results in an excessive differentiation of the crystal cell in the lymph gland. This phenotype is recapitulated by loss of Fer1HCH in the intestine, indicating that reduced levels of systemic iron enhances crystal cell differentiation. Detailed analysis of Fer1HCH-tagged-GFP revealed that Fer1HCH is also expressed in the hematopoietic systems. Lastly, blocking Fer1HCH expression in the mature blood cells showed marked increase in the blood differentiation of both crystal cells and plasmatocytes. Thus, this work suggests a relevance of systemic and local iron homeostasis in blood differentiation, prompting further investigation of molecular mechanisms underlying iron regulation and cell fate determination in the hematopoietic system (Yoo, 2017).

EFFECTS OF MUTATION

Two types of mutations in the IRE of the human L-ferritin gene cause hereditary hyperferritinemia cataract syndrome: mutations that disrupt base pairing of the stem loop and mutations that directly affect the specific CAGUG sequence of the loop and the cytosine of the IRE bulge (Cazzola 2002; Rouault 2006). The mutations that cause disease are detrimental to the defining features of IREs that are conserved from humans to flies. A survey of many sequenced Drosophila species confirmed that Fer2LCH mRNAs lack IREs, which are present only in the 5’UTR of Fer1HCH transcripts. Comparison of the new IREs identified several non-conserved nucleotides between species, but in all cases the overall structure of the stem loop remained intact and the nucleotides implicated in disease are conserved or altered in ways that are compatible with base pairing. Considering the finding that Fer2LCH mRNA lacks the IRE and is not predictably regulated by IRP-1A, it was asked whether other post-transcriptional control mechanisms govern Fer2LCH expression (Missirlis, 2007).

In order to study post-transcriptional regulation of Fer1HCH and Fer2LCH in Drosophila, flies were used with P-element insertions in these genes. Focus was placed on three P-elements: Fer1HCH⁴⁵¹ and Fer2LCH³⁵, which result in a loss-of-function mutant for each of the respective ferritin chains, and Fer2LCH^G188, which adds GFP to the H-subunit. Fer2LCH³⁵ is predicted to cause a genetic null mutation because the transposable element disrupts the open reading frame, whereas in Fer1HCH⁴⁵¹ the P-element resides in intronic sequences and the mechanism by which it disrupts gene function is unknown. When homozygous, these insertions are embryonic lethal. Each insertion present over a deficiency chromosome lacking both genes also resulted in embryonic lethality, indicating that both ferritin subunit chains perform essential functions in Drosophila (Missirlis, 2007).

To investigate possible homeostatic interactions between the two proteins, their expression in heterozygous adult flies was examined by Western blot analysis. As expected, Fer1HCH levels were markedly reduced in Fer1HCH^451/+ heterozygotes and the same was true for Fer2LCH levels in Fer2LCH^35/+ flies. Unexpectedly, endogenous levels of Fer2LCH were also low in Fer1HCH^451/+, and Fer1HCH levels were low in Fer2LCH^35/+ flies. Because trans-heterozygous (Fer1HCH⁴⁵¹/Fer2LCH³⁵) flies are fully viable, it was hypothesized that neither of these insertions would directly affect the transcriptional activity of both genes simultaneously (Missirlis, 2007).

To address the potential role of the P-element insertions on transcription at the locus, Fer1HCH was ubiquitously overexpressed in Fer1HCH⁴⁵¹/451 homozygous flies and adult viability was restored. When Fer1HCH was overexpressed in homozygous Fer2LCH³⁵/35 flies, viability was not restored. The converse was true for overexpression of Fer2LCH. These results indicate that the respective P-elements interfere specifically with Fer1HCH and Fer2LCH expression and that reduction in levels of the alternate chain in each mutant is most likely due to post-transcriptional regulation (Missirlis, 2007).

Flies were generated that express Fer1HCH with a mutation that inactivates the ferroxidase activity, based on analogy to the ferroxidase-null human allele (Cozzi, 2000). Expression of the mutated transgene was not able to functionally substitute for Fer1HCH and rescue the lethality of Fer1HCH⁴⁵¹/451 flies. Thus, the ferroxidase activity of Fer1HCH provides an essential function in vivo and is likely required for iron loading of the ferritin shell (Missirlis, 2007).

Iron deficiency reduces synapse formation in the Drosophila clock circuit

Iron serves as a critical cofactor for proteins involved in a host of biological processes. In most animals, dietary iron is absorbed in enterocytes and then disseminated for use in other tissues in the body. The brain is particularly dependent on iron. Altered iron status correlates with disorders ranging from cognitive dysfunction to disruptions in circadian activity. The exact role iron plays in producing these neurological defects, however, remains unclear. Invertebrates provide an attractive model to study the effects of iron on neuronal development since many of the genes involved in iron metabolism are conserved, and the organisms are amenable to genetic and cytological techniques. This study has examined synapse growth specifically under conditions of iron deficiency in the Drosophila circadian clock circuit. This study shows that projections of the small ventrolateral clock neurons to the protocerebrum of the adult Drosophila brain are significantly reduced upon chelation of iron from the diet. This growth defect persists even when iron is restored to the diet. Genetic neuronal knockdown of ferritin 1 or ferritin 2, critical components of iron storage and transport, does not affect synapse growth in these cells. Together, these data indicate that dietary iron is necessary for central brain synapse formation in the fly and further validate the use of this model to study the function of iron homeostasis on brain development (Rudisill, 2018).

EVOLUTIONARY HOMOLOGS

Ferritin structure

Ferritins are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. This study reports the 1.9Å structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide crosslinks reflect adaptations of insect ferritin to its role as a secreted protein (Hamburger, 2005).

Iron uptake by ferritin

Ferritin concentrates iron as a hydrous ferric oxide in a protein cavity (8 nm in diameter) by using eight pores along the threefold symmetry axes of the octahedral supramolecular structure. The role of ligand exchange in the entry of Fe(II) hexahydrate into ferritin protein has been studied with [Cr(TREN)(H(2)O)(OH)](2+) [TREN = N(CH(2)CH(2)NH(2))(3)], a model for Fe(H(2)O)(6)2+ with only two exchangeable ligands. The results show that five different ferritin proteins, varying in pore structure, oxidation sites, and nucleation sites, bind Cr(TREN) at functional protein sites, based on inhibition of iron mineralization and oxidation. Properties of Cr(TREN)-ferritin adducts include an increased isoelectric point, a shift in the Cr(TREN) UV/vis spectrum consistent with exchange of water for protein carboxylate or thiolate ligands, binding affinities of 50-250 microM, and a slow rate of dissociation (k = 4 x 10(-6) sec(-1)). The relationship of Cr(TREN) inhibition of iron oxidation and mineralization by Cr(TREN) to the known structures of the various ferritins tested suggests that Cr(TREN) plugs the ferritin pores, obstructing Fe(II) entry in folded and unfolded pores. Because only two exchangeable waters are sufficient for pore binding of Cr(TREN), the physiological Fe(II) donor must bind to the pore with few exchangeable ligands. These results show the advantage of using stable model complexes to explore properties of transient Fe-protein complexes during Fe mineralization in ferritin (Barnes, 2002).

Translational regulation of Ferritin RNA; Iron regulatory protein function

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. This study generated mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases (Lavaute, 2001).

In mammals, iron regulatory proteins 1 and 2 (IRP1 and IRP2) posttranscriptionally regulate expression of several iron metabolism proteins including ferritin and transferrin receptor. Genetically engineered mice that lack IRP2, but have the normal complement of IRP1, develop adult-onset neurodegenerative disease associated with inappropriately high expression of ferritin in degenerating neurons. Mice that are homozygous for a targeted deletion of IRP2 and heterozygous for a targeted deletion of IRP1 (IRP1+/- IRP2-/-) develop a much more severe form of neurodegeneration, characterized by widespread axonopathy and eventually by subtle vacuolization in several areas, particularly in the substantia nigra. Axonopathy develops in white matter tracts in which marked increases in ferric iron and ferritin expression are detected. Axonal degeneration is significant and widespread before evidence for abnormalities or loss of neuronal cell bodies can be detected. Ultimately, neuronal cell bodies degenerate in the substantia nigra and some other vulnerable areas, microglia are activated, and vacuoles appear. Mice manifest gait and motor impairment at stages when axonopathy is pronounced, but neuronal cell body loss is minimal. These observations suggest that therapeutic strategies that aim to revitalize neurons by treatment with neurotrophic factors may be of value in IRP2-/- and IRP1+/- IRP2-/- mouse models of neurodegeneration (Smith, 2004).

Iron-regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2-/- mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. This study reports that ablation of IRP2 results in iron-limited erythropoiesis. TfR expression in erythroid precursors of IRP2-/- mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRP-dependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2-/- erythroid cells. IRP2-/- mice represent a new paradigm of genetic microcytic anemia. It is postulated that IRP2 mutations or deletions may be a cause of refractory microcytic anemia and bone marrow iron depletion in patients with normal transferrin saturations, elevated serum ferritins, elevated red cell protoporphyrin IX levels, and adult-onset neurodegeneration (Cooperman, 2005).

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. Young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1 (Galy, 2005).

The acute box cis-element in human heavy ferritin mRNA 5'-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins

Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5'-untranslated region. Iron-responsive element (IRE). Iron and interleukin-1beta (IL-1beta) act synergistically to increase H- and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5'-untranslated region, conferred a substantial increase in basal and IL-1beta-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-alphaCP1 and -alphaCP2, which have well defined roles as translation regulators. Iron influx increased the association of alphaCP1 with ferritin mRNA and decreased the alphaCP2-ferritin mRNA interaction, whereas IL-1beta reduced the association of alphaCP1 and alphaCP2 with H-ferritin mRNA. In summary, the H-ferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver (Thomson, 2005).

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 Å resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by approximately 30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme (Walden, 2006).

Mutation and ectopic expression of Ferritin

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H₂O₂ toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data (Cozzi, 2000).

Ferritin, the iron-storing molecule, is made by the assembly of various proportions of 2 different H and L subunits into a 24-mer protein shell. These heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. H ferritin is indispensable for normal development, since inactivation of the H ferritin gene by homologous recombination in mice is lethal at an early stage during embryonic development. The phenotypic analysis of the mice heterozygous for the H ferritin gene (Fth+/- mice) is reported in this study, and differences in gene regulation between the 2 subunits are shown. The heterozygous Fth+/- mice were healthy and fertile and did not present any apparent abnormalities. Although they had iron-overloaded spleens at the adult stage, this is identical to what is observed in normal Fth+/+ mice. However, these heterozygous mice had slightly elevated tissue L ferritin content and 7- to 10-fold more L ferritin in the serum than normal mice, but their serum iron remained unchanged. H ferritin synthesis from the remaining allele was not up-regulated. This probably results from subtle changes in the intracellular labile iron pool, which would stimulate L ferritin but not H ferritin synthesis. These results raise the possibility that reduced H ferritin expression might be responsible for unexplained human cases of hyperferritinemia in the absence of iron overload where the hereditary hyperferritinemia-cataract syndrome has been excluded (Ferreira, 2001).

The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, it was determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). Differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. It is concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain (Goralska, 2003).

Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. A heavy chain (H) ferritin null mutant was developed in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. Heterozygous and wild-type (wt) mice were examined between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, it is hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (SOD) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-3 were detected in neurons of the +/- mice but not in the wt. Thus, a mouse model was developed that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies (Thompson, 2003).

Small interfering RNAs (siRNAs) were used to down-regulate H- and L-ferritin levels in HeLa cells. siRNAs repressed H- and L-ferritin expression to about 20% to 25% of the background level in both stable and transient transfections. HeLa cells transfected with H- and L-ferritin cDNAs were analyzed in parallel to compare the effects of ferritin up- and down-regulation. Large modifications of L-ferritin levels did not affect iron availability in HeLa cells but positively affected cell proliferation rate in an iron-independent manner. The transient down-regulation of H-ferritin modified cellular iron availability and resistance to oxidative damage, as expected. In contrast, the stable suppression of H-ferritin in HeLa cell clones transfected with siRNAs did not increase cellular iron availability but made cells less resistant to iron supplementation and chelation. The results indicate that L-ferritin has no direct effects on cellular iron homeostasis in HeLa cells, while it has new, iron-unrelated functions. In addition, they suggest that H-ferritin function is to act as an iron buffer (Cozzi, 2004).

Ferritin is an iron storage protein made of 24 subunits. Previous mutational analyses showed that ferritin C-terminal region has a major role in protein stability and assembly but is only marginally involved in the mechanism of iron incorporation. However, it has recently been shown that patients who carry alterations of ferritin C-terminal sequence caused by nucleotide insertions show neurological disorders possibly related to altered protein functionality and cellular iron deregulation. To re-evaluate the role of this region, five mutants of mouse H-ferritin were produced by 2-nucleotide insertions that modified the last 6-29 residues and extended the sequence of 14 amino acids. The mutants were expressed in Escherichia coli and analysed for solubility, stability and capacity to incorporate iron. The alteration of the last 6-residue non-helical extension had no evident effect on the properties of ferritin, while solubility and capacity to assemble in ferritin shells decreased progressively with the extension of the modified region. The results also showed that the modification of even a part of the terminal E-helix abolished the capacity of ferritin to incorporate iron during expression in the cells, probably caused by conformational modification of the hydrophobic channels. The data support the hypothesis that the pathogenic mutations alter cellular iron homeostasis (Ingrassia, 2006).

Transcriptonal regulation of Ferritin

A major enhancer of the mouse ferritin H gene (FER-1) is central to repression of the ferritin H gene by the adenovirus E1A oncogene. To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that fail to bind p300/CBP loose the ability to repress FER-1, whereas mutants of E1A that abrogate its interaction with Rb, p107, or p130 are fully functional in transcriptional repression. Transfection with E1A does not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, it has been demonstrated that transfection of a p300 expression plasmid significantly activates ferritin H-CAT containing the FER-1 enhancer, but has a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that fail to bind E1A but retain an adaptor function restore FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimics p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP (Tsuji, 1999).

Ferritin is a ubiquitous intracellular iron storage protein that consists of 24 subunits of the H and L type. The ability to sequester iron from participation in oxygen free radical formation is consistent with a cytoprotective role for ferritin. This study demonstrates that ferritins H and L are induced in cells treated with β-napthoflavone (β-NF) and chemopreventive dithiolethiones. Induction of ferritin H by β-NF and the dithiolethiones oltipraz and 1,2-dithiole-3-thione (D3T) occurs via a transcriptional mechanism that is mediated by the ferritin H electrophile/antioxidant-responsive element (EpRE/ARE). The murine ferritin H gene contains five potential xenobiotic-responsive element (XRE) sequences in its 5'-promoter region. However, deletion analysis demonstrates that these XRE sequences are not functional in inducing ferritin H in response to β-NF. Electrophoretic mobility shift assays demonstrate that the ferritin H EpRE/ARE binds Nrf2. Transfection of chimeric ferritin H reporter genes with Nrf2 expression vectors and Nrf2 dominant-negative mutants indicate that Nrf2 functions at the EpRE/ARE to mediate transcriptional activation of ferritin H. Induction of ferritin H and L was not seen in Nrf2 knockout cells, demonstrating that this transcription factor is required for the induction of ferritin in response to polycyclic aromatic xenobiotics and chemopreventive agents. Nrf2 may also play a role in basal transcription of both ferritin H and L. These results provide a mechanistic link between regulation of the iron storage protein ferritin and the cancer chemopreventive response (Pietsch, 2003).

During inflammation, NF-kappaB transcription factors antagonize apoptosis induced by tumor necrosis factor (TNF)alpha. This antiapoptotic activity of NF-kappaB involves suppressing the accumulation of reactive oxygen species (ROS) and controlling the activation of the c-Jun N-terminal kinase (JNK) cascade. However, the mechanism(s) by which NF-kappaB inhibits ROS accumulation is unclear. Ferritin heavy chain (FHC), the primary iron storage factor, has been identified as an essential mediator of the antioxidant and protective activities of NF-kappaB. FHC is induced downstream of NF-kappaB and is required to prevent sustained JNK activation and, thereby, apoptosis triggered by TNFalpha. FHC-mediated inhibition of JNK signaling depends on suppressing ROS accumulation and is achieved through iron sequestration. These findings establish a basis for the NF-kappaB-mediated control of ROS induction and identify a mechanism by which NF-kappaB suppresses proapoptotic JNK signaling. These results suggest modulation of FHC or, more broadly, of iron metabolism as a potential approach for anti-inflammatory therapy (Pham, 2004).

Ferritin is the major intracellular iron storage protein that sequesters excess free iron to minimize generation of iron-catalysed reactive oxygen species. Expression of ferritin heavy chain (ferritin H) is induced by pro-oxidants as part of a cellular antioxidant response to protect cells from oxidative damage. The antioxidant/electrophile response element (ARE) located 4.5 kb upstream to the human ferritin H transcription initiation site is responsible for the oxidant response. The human ferritin H ARE comprises two copies of bidirectional AP1 motifs. Mutations in each AP1 motif significantly impairs protein binding and the function of the ARE, indicating that both of the AP1 motifs are required for pro-oxidant-mediated activation of the ferritin H gene. JunD, an AP1 family basic-leucine zipper (bZip) transcription factor, is one of the ferritin H ARE binding proteins and activates ferritin H transcription in HepG2 hepatocarcinoma cells. Gel retardation assay demonstrated that H2O2 (hydrogen peroxide) or t-BHQ (tert-butylhydroquinone) treatment increases total protein binding as well as JunD binding to the ferritin H ARE. Chromatin immunoprecipitation assay showed that H2O2 treatment induces JunD binding to the ferritin H ARE. Both H2O2 and t-BHQ induce phosphorylation of JunD at Ser-100, an activated form of JunD. Furthermore, overexpression of JunD induces endogenous ferritin H protein synthesis. Since JunD has been demonstrated to protect cells from several stress stimuli including oxidative stress, these results suggest that, in addition to NFE2-related factor 2 (Nrf2) as a major ARE regulatory protein, JunD is another ARE regulatory protein for transcriptional activation of the human ferritin H gene and probably other antioxidant genes containing the conserved ARE sequences by which JunD may confer cytoprotection during oxidative stress (Tsuji, 2005).

An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. This study found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5' regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE (Iwasaki, 2006).

Gene transcription is coordinately regulated by the balance between activation and repression mechanisms in response to various external stimuli. Ferritin, composed of H and L subunits, is the major intracellular iron storage protein involved in iron homeostasis. An enhancer, termed antioxidant responsive element (ARE), has been identified in the human ferritin H gene and its respective transcriptional activators including Nrf2 and JunD. This study found that ATF1 (activating transcription factor 1) is a transcriptional repressor of the ferritin H ARE. Subsequent yeast two-hybrid screening identified PIAS3 (protein inhibitor of activated STAT3) as an ATF1 binding protein. Further investigation of the human ferritin H ARE regulation showed that (1) PIAS3 reversed ATF1-mediated repression of the ferritin H ARE, (2) ATF1 was sumoylated, but PIAS3, a SUMO E3 ligase, did not appear to play a major role in SUMO1-mediated ATF1 sumoylation or ATF1 transcription activating function, (3) PIAS3 decreased ATF1 binding to the ARE, and (4) ATF1 knockdown with siRNA increased ferritin H expression, while PIAS3 knockdown decreased basal expression and oxidative stress-mediated induction of ferritin H. These results suggest that PIAS3 antagonizes the repressor function of ATF1, at least in part by blocking its DNA binding, and ultimately activates the ARE. Collectively these results suggest that PIAS3 is a new regulator of ATF1 that regulates the ARE-mediated transcription of the ferritin H gene (Iwasaki, 2007).

Ferritin subcellular distribution

This study characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species by sequestering iron in the mature erythrocyte. Moreover, the data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir (Infante, 2007).

Secretion of Ferritin

Serum ferritin has been used widely in clinical medicine chiefly as an indicator of iron stores and inflammation. Circulating ferritin also can have paracrine effects. Despite the clinical significance of serum ferritin, its secretion remains an enigma. The consensus view is that serum ferritin arises from tissue ferritins -- principally ferritin light -- which can be glycosylated. Ferritin heavy and light chains are cytosolic proteins that form cages of 24 subunits to store intracellular iron. Ferritin light is secreted when its expression is increased in stable, transfected HepG2 cells or adenovirus-infected HepG2 cells. Export occurs through the classical secretory pathway and some chains are N-glycosylated. Ferritins do not need to form cages prior to secretion. Secretion is blocked specifically, effectively, and rapidly by a factor in serum. The timing of this inhibition of ferritin secretion suggests that normally cytosolic ferritin L is targeted to the secretory pathway during translation despite the absence of a conventional signal sequence. Thus, secretion of glycosylated and unglycosylated ferritin is a regulated and not a stochastic process (Ghosh, 2004).

Tissue-specific expression of ferritin H regulates cellular iron homoeostasis

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, transgenic mice were prepared that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0-fold compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2-fold. Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3-fold, concurrent with a 4.5-fold increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance (Wilkinson, 2006).

Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance

Secreted ferritin in the mosquito, Aedes aegypti, has several subunits that are the products of at least two genes, one encoding a homologue of the vertebrate heavy chain (HCH) and the other the light chain homologue (LCH). This study reports the developmental and organ specific pattern of expression of the ferritin HCH messages and of both subunit types in control sugar-fed mosquitoes, in those exposed to high levels of dietary iron, and after blood feeding. When Northern blots were probed with a HCH cDNA, two bands were observed, representing at least two messages of different sizes that result from the choice of two different polyadenylation sites. Either raising mosquito larvae in an iron-enriched medium, or blood feeding adult female mosquitoes resulted in a marked increase in the HCH message level, particularly of the shorter message. Changes in the amount and length of messages and amount of ferritin subunits were studied over the life span of the mosquito and in different organs of female mosquitoes after blood feeding. The midgut of blood-fed insects is the main site of increased ferritin message synthesis. Ferritin protein levels also increase in midgut, fat body and hemolymph after blood feeding. Ferritin messages and subunits are synthesized in the ovaries and ferritin is found in the eggs. These observations are discussed in terms of translational and transcriptional control of ferritin synthesis and are compared to similar events in the regulation of Drosophila ferritin (Dunkov, 2002).

Natural blood meals are rarely enriched in non-heme iron, but they are very rich in heme, a potent prooxidant. The results presented in this study indicate that ferritin, which accumulates in female mosquitoes after a blood meal, probably serves a dual function: 1) to sequester iron released from heme and thus prevent oxidative damage; and 2) to provide an iron store in the eggs to be used by the developing embryos. The role of ferritin as an antioxidant in various mammalian tissues is well established. The current results, demonstrating that the midgut of blood-fed mosquitoes is a major site of ferritin message and subunit synthesis, are consistent with such antioxidant role of ferritin (Dunkov, 2002).

The dramatic increase of ferritin HCH messages in the midgut after a blood meal could be the source of ferritin in the hemolymph. This is supported by studies with Drosophila where high hemolymph ferritin concentrations have also been attributed to increased ferritin message synthesis in the midgut (Georgieva, 2002). Secretion of ferritin from the fat body could also contribute to the hemolymph ferritin pool, as suggested by the increased ferritin abundance in this tissue after a blood meal. A portion of the midgut ferritin in insects might be secreted into the lumen. However, as the peritrophic matrix is not permeable for ferritin any ferritin that enters the lumen would remain between the gut wall and peritrophic matrix. The results indicate that no detectable amounts of excreted ferritin were present in the midgut 24h after a blood meal. Consistent with these results, ferritin was not detected in extracts from whole hindguts 24h after a blood meal (Dunkov, 2002).

It is interesting that a protein band of ~30 kDa, most likely corresponding to unprocessed HCH or LCH ferritin polypeptides, is seen on Western blots prepared from both midgut and ovaries. This is consistent with the increased ferritin HCH message abundance in these organs observed after a blood meal, which probably results in elevated ferritin synthesis and thus allows detection of unprocessed polypeptides. Interestingly, a 30 kDa band was observed in eggs with pharate 1st instar larvae as well, but not in early embryos. Most likely, ferritin synthesis is low during early embryogenesis but resumes in pharate 1st instar larvae, resulting in the detection of the 30 kDa band. The observed accumulation of ferritin in the ovaries and eggs of Ae. aegypti is in accord with similar findings in D. melanogaster (Georgieva, 2002). These results are also in agreement with studies on Sarcophaga peregrina suggesting that ferritin is the main iron storage protein in the eggs of this fly. In addition, the presence of ferritin in the yolk and in the vitelline membrane of the oocyte in the moth Galleria mellonella has been demonstrated. Ferritin is also the major yolk protein in snails and it is found in the eggs of amphibians (Dunkov, 2002).

It is noteworthy that in contrast to D. melanogaster, HCH is the predominant ferritin subunit type in Ae. aegypti. While the roles of HCH and LCH chains in the function of insect secreted ferritins are not clear, the observed differences might reflect specific iron storage requirements of these insects. Indeed, Drosophila is rarely exposed to high iron diets while Ae. aegypti females take blood meals rich in heme iron. HCH ferritin chains may be somehow more effective in meeting the increased need for iron sequestration after a blood meal (Dunkov, 2002).

In conclusion, the results describing the constitutive and inducible temporal and spatial pattern of ferritin expression provide a background for further investigation of the molecular mechanisms that control ferritin function in the mosquito Ae. aegypti. Two observations deserve special attention: the transient upregulation of ferritin HCH gene and the accumulation of secreted ferritin in the hemolymph after a natural blood meal. Together with the already known gene structure, and with the available technology for producing transgenic mosquitoes, these results should stimulate studies designed to use the ferritin HCH gene promoter for directing expression of antiparasite genes in a time and tissue specific manner (Dunkov, 2002).

Ferritin and neurodegeneration

Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. This study explored the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson's-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in these studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease (Kaur, 2003).

Ferritin elevation has been reported by some laboratories to occur within the substantia nigra (SN), the area of the brain affected in Parkinson's disease (PD), but whether such an increase could be causatively involved in neurodegeneration associated with the disorder is unknown. This study reports that chronic ferritin elevation in midbrain dopamine-containing neurons results in a progressive age-related neurodegeneration of these cells. This provides strong evidence that chronic ferritin overload could be directly involved in age-related neurodegeneration such as occurs in Parkinson's and other related diseases (Kaur, 2006).

Ferritin is required in multiple tissues during Drosophila melanogaster development

In Drosophila, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. This study presents analyses of embryonic phenotypes for mutations in Fer1HCH, Fer2LCH and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, it was shown that ferritin maternal contribution, which varies reflecting the mother's iron stores, is used in early development. The study also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, these results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during Drosophila embryonic development (González-Morales, 2015).

REFERENCES

Search PubMed for articles about Drosophila Ferritin

Barnes, C. M., Theil, E. C. and Raymond, K. N. (2002). Iron uptake in ferritin is blocked by binding of [Cr(TREN)(H(2)O)(OH)](2+), a slow dissociating model for [Fe(H(2)O)(6)](2+). Proc. Natl. Acad. Sci. 99(8): 5195-200. Medline abstract: 11959967

Beutler, E., et al. (2002). Relationship of body iron stores to levels of serum ferritin, serum iron, unsaturated iron binding capacity and transferrin saturation in patients with iron storage disease. Acta Haematol 107: 145-149. Medline abstract: 11978935

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Cozzi, A., et al. (2000). Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J. Biol. Chem. 275: 25122-25129. Medline abstract: 10833524

Cozzi, A., et al. (2004). Analysis of the biologic functions of H- and L-ferritins in HeLa cells by transfection with siRNAs and cDNAs: evidence for a proliferative role of L-ferritin. Blood 103(6): 2377-83. Epub 2003 Nov 13. Medline abstract: 14615379

Dunkov, B. C. and Georgieva, T. (1999). Organization of the ferritin genes in Drosophila melanogaster. DNA Cell Biol. 18(12): 937-44. Medline abstract: 10619605

Dunkov, B. C., Georgieva, T., Yoshiga, T., Hall, M. and Law, J. H. (2002). Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J. Insect Sci. 2: 7. Medline abstract: 15455041

Dunkov, B., and Georgieva, T. (2006). Insect iron binding proteins: insights from the genomes. Insect Biochem. Mol. Biol. 36: 300-309. Medline abstract: 16551544

Ferreira, C., et al. (2001). H ferritin knockout mice: a model of hyperferritinemia in the absence of iron overload. Blood 98(3): 525-32. Medline abstract: 11468145

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Georgieva, T., Dunkov, B. C., Harizanova, N., Ralchev, K. and Law, J. H. (1999). Iron availability dramatically alters the distribution of ferritin subunit messages in Drosophila melanogaster. Proc. Natl. Acad. Sci. 96(6): 2716-21. Medline abstract: 10077577 [PubMed - indexed for MEDLINE]

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Ghosh, S., Hevi, S. and Chuck, S. L. (2004). Regulated secretion of glycosylated human ferritin from hepatocytes. Blood 103: 2369-2376. Medline abstract: 14615366

González-Morales, N., Mendoza-Ortíz, M.Á., Blowes, L.M., Missirlis, F. and Riesgo-Escovar, J.R. (2015). Ferritin is required in multiple tissues during Drosophila melanogaster development. PLoS One 10: e0133499. PubMed ID: 26192321

Goralska, M., Holley, B. L. and McGahan, M. C. (2003). Identification of a mechanism by which lens epithelial cells limit accumulation of overexpressed ferritin H-chain. J. Biol. Chem. 278(44): 42920-6. Medline abstract: 12920121

Gray, N. K., Pantopoulos, K., Dandekar, T., Ackrell, B. A. and Hentze, M. W. (1996). Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc. Natl. Acad. Sci. 93(10): 4925-30. Medline abstract: 8643505

Hamburger, A. E., et al. (2005). Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains. J. Mol. Biol. 349: 558-569. Medline abstract: 15896348

Infante, A. A., et al. (2007). Ferritin associates with marginal band microtubules. Exp. Cell Res. 313(8): 1602-14. Medline abstract: 17391669

Ingrassia, R., Gerardi, G., Biasiotto, G. and Arosio, P. (2006). Mutations of ferritin H chain C-terminus produced by nucleotide insertions have altered stability and functional properties. J. Biochem. (Tokyo). 139(5): 881-5. Medline abstract: 16751596

Iwasaki, K., et al. (2006). Hemin-mediated regulation of an antioxidant-responsive element of the human ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells. Mol. Cell. Biol. 26(7):2845-56. Medline abstract: 16537925

Iwasaki, K., Hailemariam, K. and Tsuji, Y. (2007). Protein inhibitor of activated STAT3 (PIAS3) interacts with activating transcription factor 1 (ATF1) and regulates the human ferritin H gene through an antioxidant responsive element. J. Biol. Chem. 282(31): 22335-43. Medline abstract: 17565989

Kaur, D., et al. (2003). Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease. Neuron 37: 899-909. Medline abstract: 12670420

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date revised: 7 August 2020

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