Ferritins: iron/oxygen biominerals in protein nanocages

Ferritin protein nanocages that form iron oxy biominerals in the central nanometer cavity are nature’s answer to managing iron and oxygen; gene deletions are lethal in mammals and render bacteria more vulnerable to host release of antipathogen oxidants. The multifunctional, multisubunit proteins couple iron with oxygen (maxi-ferritins) or hydrogen peroxide (mini-ferritins) at catalytic sites that are related to di-iron sites oxidases, ribonucleotide reductase, methane monooxygenase and fatty acid desaturases, and synthesize mineral precursors. Gated pores, distributed symmetrically around the ferritin cages, control removal of iron by reductants and chelators. Gene regulation of ferritin, long known to depend on iron and, in animals, on a noncoding messenger RNA (mRNA) structure linked in a combinatorial array to functionally related mRNA of iron transport, has recently been shown to be linked to an array of proteins for antioxidant responses such as thioredoxin and quinone reductases. Ferritin DNA responds more to oxygen signals, and ferritin mRNA responds more to iron signals. Ferritin genes (DNA and RNA) and protein function at the intersection of iron and oxygen chemistry in biology.     

Coordinating responses to iron and oxygen stress with DNA and mRNA promoters: The ferritin story

Combinations of DNA antioxidant response element and mRNA iron responsive element regulate ferritin expression in animals in response to oxidant and iron stress, or normal developmental signals. Ferritins are protein nanocages, found in animals, plants, bacteria, and archaea, that convert iron and oxygen to ferric oxy biominerals in the protein central cavity; the mineral traps potentially toxic reactants and concentrates iron for the future synthesis of other iron/heme proteins. Regulatory signals and the nanocage gene products are the same throughout biology, but the genetic mechanisms, DNA versus DNA + mRNA, vary. The number of genes, temporal regulation, tissue distribution in multi-cellular organisms, and gene product size (maxi-ferritins have 24 subunits and mini-ferritins, or Dps proteins, have 12 subunits and are restricted to bacteria and archaea) suggest an overwhelming diversity and variability. However, common themes of regulation and function are described which indicate not only that the three-dimensional protein structure and the functions of the ferritins are conserved, but also that broad features of genetic regulation are conserved relative to organismal and/or community needs. The analysis illustrates the centrality of the ferritins to life with iron and oxygen and models how Nature harnesses potentially dangerous chemistry for biology.     

Ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry

The ferritin superfamily is composed of ancient, nanocage proteins with an internal cavity, 60% of total volume, that reversibly synthesize solid minerals of hydrated ferric oxide; the minerals are iron concentrates for cell nutrition as well as antioxidants due to ferrous and oxygen consumption during mineralization. The cages have multiple iron entry/exit channels, oxidoreductase enzyme sites, and, in eukaryotes, Fe(III)O nucleation channels with clustered exits that extend protein activity to include facilitated mineral growth. Ferritin protein cage differences include size, amino acid sequence, and location of the active sites, oxidant substrate and crystallinity of the iron mineral. Genetic regulation depends on iron and oxygen signals, which in animals includes direct ferrous signaling to RNA to release and to ubiquitin-ligases to degrade the protein repressors. Ferritin biosynthesis forms, with DNA, mRNA and the protein product, a feedback loop where the genetic signals are also protein substrates. The ferritin protein nanocages, which are required for normal iron homeostasis and are finding current use in the delivery of nanodrugs, novel nanomaterials, and nanocatalysts, are likely contributors to survival and success during the transition from anaerobic to aerobic life.     

Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

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

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

Molecular characterization of the iron binding protein ferritin in Eisenia andrei earthworms

Ferritin is a storage protein that plays a key role in iron metabolism. In this study, we report on the sequence characterization of a ferritin-coding cDNA in Eisenia andrei earthworms isolated by RT-PCR using degenerated primers, and we suggest the presence of a putative IRE in the 5′-UTR of ferritin mRNA. The obtained ferritin sequence was compared with those of other animals showing sequence and structure homology in consensus sites, including the iron-responsive element (IRE) and ferroxidase centers. Despite the sequence homology in the E. andrei mRNA of ferritin with the sequences of other animals in consensus IRE sites, the presented cytosine in the IRE of E. andrei ferritin in the expected position does not form a conventional bulge. The presence of ferritin in the coelomic fluid of E. andrei was proven by iron staining assay. Moreover, aconitase activity in the coelomic fluid was assessed by aconitase assay, suggesting the presence of an iron regulatory protein. Quantitative analysis revealed changes in the gene expression levels of ferritin in coelomocytes in response to bacterial challenge, reaching the maximum level 8 h after the stimulation with both Gram-positive and Gram-negative bacteria.     

Cytoprotective Effect of Ferritin H in Renal Ischemia Reperfusion Injury

Oxidative stress is a major contributor to kidney injury following ischemia reperfusion. Ferritin, a highly conserved iron-binding protein, is a key protein in the maintenance of cellular iron homeostasis and protection from oxidative stress. Ferritin mitigates oxidant stress by sequestering iron and preventing its participation in reactions that generate reactive oxygen species. Ferritin is composed of two subunit types, ferritin H and ferritin L. Using an in vivo model that enables conditional tissue-specific doxycycline-inducible expression of ferritin H in the mouse kidney, we tested the hypothesis that an increased level of H-rich ferritin is renoprotective in ischemic acute renal failure. Prior to induction of ischemia, doxycycline increased ferritin H in the kidneys of the transgenic mice nearly 6.5-fold. Following reperfusion for 24 hours, induction of neutrophil gelatinous-associated lipocalin (NGAL, a urine marker of renal dysfunction) was reduced in the ferritin H overexpressers compared to controls. Histopathologic examination following ischemia reperfusion revealed that ferritin H overexpression increased intact nuclei in renal tubules, reduced the frequency of tubular profiles with luminal cast materials, and reduced activated caspase-3 in the kidney. In addition, generation of 4-hydroxy 2-nonenal protein adducts, a measurement of oxidant stress, was decreased in ischemia-reperfused kidneys of ferritin H overexpressers. These studies demonstrate that ferritin H can inhibit apoptotic cell death, enhance tubular epithelial viability, and preserve renal function by limiting oxidative stress following ischemia reperfusion injury.     

The molecular cloning and characterization of murine ferritin heavy chain, a tumor necrosis factor-inducible gene

Ferritin is a ubiquitous and highly conserved protein which plays a major role in iron homeostasis. We have identified and sequenced a full-length cDNA for murine ferritin heavy chain. The isolated cDNA is 819 nucleotides in length. It includes 546 nucleotides which encode a protein of 182 amino acids, a 5' noncoding sequence of 120 nucleotides, and a 3'-noncoding region of 153 nucleotides. The sequence displays a high degree of homology to human ferritin H, and includes a portion of the iron-responsive element conserved in chick, frog, and human ferritin. Tumor necrosis factor (TNF), a cytokine which mediates elements of the stress response, induces expression of ferritin H mRNA. Both mouse TA1 adipocytes and human muscle cells increase expression of ferritin H mRNA 4-6-fold after 48 h exposure to TNF. This increase occurs both prior and subsequent to differentiation of adipocytes and muscle cells, and is accompanied by an increase in the synthesis of the ferritin H subunit. These findings suggest a novel role for TNF in iron metabolism.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)