Ferritins: furnishing proteins with iron

Ferritins are a superfamily of iron oxidation, storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. The majority of ferritins consist of 24 subunits that individually fold into 4-α-helix bundles and assemble in a highly symmetric manner to form an approximately spherical protein coat around a central cavity into which an iron-containing mineral can be formed. Channels through the coat at inter-subunit contact points facilitate passage of iron ions to and from the central cavity, and intrasubunit catalytic sites, called ferroxidase centers, drive Fe2+ oxidation and O2 reduction. Though the different members of the superfamily share a common structure, there is often little amino acid sequence identity between them. Even where there is a high degree of sequence identity between two ferritins there can be major differences in how the proteins handle iron. In this review we describe some of the important structural features of ferritins and their mineralized iron cores and examine in detail how three selected ferritins oxidise Fe2+ in order to explore the mechanistic variations that exist amongst ferritins. We suggest that the mechanistic differences reflect differing evolutionary pressures on amino acid sequences, and that these differing pressures are a consequence of different primary functions for different ferritins

Mathematical modeling of the dynamic storage of iron in ferritin

<p>Abstract</p> <p>Background</p> <p>Iron is essential for the maintenance of basic cellular processes. In the regulation of its cellular levels, ferritin acts as the main intracellular iron storage protein. In this work we present a mathematical model for the dynamics of iron storage in ferritin during the process of intestinal iron absorption. A set of differential equations were established considering kinetic expressions for the main reactions and mass balances for ferritin, iron and a discrete population of ferritin species defined by their respective iron content.</p> <p>Results</p> <p>Simulation results showing the evolution of ferritin iron content following a pulse of iron were compared with experimental data for ferritin iron distribution obtained with purified ferritin incubated <it>in vitro </it>with different iron levels. Distinctive features observed experimentally were successfully captured by the model, namely the distribution pattern of iron into ferritin protein nanocages with different iron content and the role of ferritin as a controller of the cytosolic labile iron pool (cLIP). Ferritin stabilizes the cLIP for a wide range of total intracellular iron concentrations, but the model predicts an exponential increment of the cLIP at an iron content > 2,500 Fe/ferritin protein cage, when the storage capacity of ferritin is exceeded.</p> <p>Conclusions</p> <p>The results presented support the role of ferritin as an iron buffer in a cellular system. Moreover, the model predicts desirable characteristics for a buffer protein such as effective removal of excess iron, which keeps intracellular cLIP levels approximately constant even when large perturbations are introduced, and a freely available source of iron under iron starvation. In addition, the simulated dynamics of the iron removal process are extremely fast, with ferritin acting as a first defense against dangerous iron fluctuations and providing the time required by the cell to activate slower transcriptional regulation mechanisms and adapt to iron stress conditions. In summary, the model captures the complexity of the iron-ferritin equilibrium, and can be used for further theoretical exploration of the role of ferritin in the regulation of intracellular labile iron levels and, in particular, as a relevant regulator of transepithelial iron transport during the process of intestinal iron absorption.</p

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

Molecular Cloning and Copy Number Variation of a Ferritin Subunit (Fth1) and Its Association with Growth in Freshwater Pearl Mussel Hyriopsis cumingii

Iron is one of the most important minor elements in the shells of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell growth. A novel ferritin subunit (Fth1) cDNA from the freshwater pearl mussel (Hyriopsis cumingii) was isolated and characterized. The complete cDNA contained 822 bp, with an open reading frame (ORF) of 525 bp, a 153 bp 5′ untranslated region (UTR) and a 144 bp 3′ UTR. The complete genomic DNA was 4125 bp, containing four exons and three introns. The ORF encoded a protein of 174 amino acids without a signal sequence. The deduced ferritin contained a highly conserved motif for the ferroxidase center comprising seven residues of a typical vertebrate heavy-chain ferritin. It contained one conserved iron associated residue (Try27) and iron-binding region signature 1 residues. The mRNA contained a 27 bp iron-responsive element with a typical stem-loop structure in the 5′-UTR position. Copy number variants (CNVs) of Fth1 in two populations (PY and JH) were detected using quantitative real-time PCR. Associations between CNVs and growth were also analyzed. The results showed that the copy number of the ferritin gene of in the diploid genome ranged from two to 12 in PY, and from two to six in JH. The copy number variation in PY was higher than that in JH. In terms of shell length, mussels with four copies of the ferritin gene grew faster than those with three copies (P<0.05), suggesting that CNVs in the ferritin gene are associated with growth in shell length and might be a useful molecular marker in selective breeding of H. cumingii

Metal–organic complexation in the marine environment

We discuss the voltammetric methods that are used to assess metal–organic complexation in seawater. These consist of titration methods using anodic stripping voltammetry (ASV) and cathodic stripping voltammetry competitive ligand experiments (CSV-CLE). These approaches and a kinetic approach using CSV-CLE give similar information on the amount of excess ligand to metal in a sample and the conditional metal ligand stability constant for the excess ligand bound to the metal. CSV-CLE data using different ligands to measure Fe(III) organic complexes are similar. All these methods give conditional stability constants for which the side reaction coefficient for the metal can be corrected but not that for the ligand. Another approach, pseudovoltammetry, provides information on the actual metal–ligand complex(es) in a sample by doing ASV experiments where the deposition potential is varied more negatively in order to destroy the metal–ligand complex. This latter approach gives concentration information on each actual ligand bound to the metal as well as the thermodynamic stability constant of each complex in solution when compared to known metal–ligand complexes. In this case the side reaction coefficients for the metal and ligand are corrected. Thus, this method may not give identical information to the titration methods because the excess ligand in the sample may not be identical to some of the actual ligands binding the metal in the sample

HIF-1 Regulates Iron Homeostasis in Caenorhabditis elegans by Activation and Inhibition of Genes Involved in Iron Uptake and Storage

Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1–dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage