Releasing iron from ferritin protein nanocage by reductive method: The role of electron transfer mediator

Background

Ferritin detoxifies excess of free Fe(II) and concentrates it in the form of ferrihydrite (Fe₂O₃·xH₂O) mineral. When in need, ferritin iron is released for cellular metabolic activities. However, the low solubility of Fe(III) at neutral pH, its encapsulation by stable protein nanocage and presence of dissolved O₂ limits in vitro ferritin iron release.

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.     

Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T ₂ and T ₂*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as “probeless” magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T ₂- and T ₂*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin’s stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T ₂ relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T ₂ relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.     

Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.     

Hemolymph ferritin and radula structure in the limpets Patelloida alticostata and Patella peronii (Mollusca: Gastropoda)

• 1.1. The mean concentration of total hemolymph iron was 3060μg/100 ml in Patella peronii and 2950μg/100 ml in Patelloida alticostata.
• 2.2. Ferritin was found to act as a major iron-binding protein in the hemolymph of both P. peronii and P. alticostata.
• 3.3. P. alticostata ferritin has a molecular weight of approximately 505,000, while that of P. peronii has a mol wt. of approximately 520,000.
• 4.4. The lateral radula teeth of both species are mineralized by deposits of silica (SiO₂) and iron in the form of goethite (α-FeOOH).
• 5.5. Hemolymph ferritin is suggested to act as a high capacity transport system to supply iron to the mineralizing front of the radula.

     

Ferritin iron mineralization proceeds by different mechanisms in MOPS and imidazole buffers

The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. Ferritin iron loading in imidazole shows a rapid hyperbolic curve in contrast to iron loading in 3-(N-morpholino)propanesulfonic acid (MOPS), which displays a slower sigmoidal curve. Ferritin iron loading in an equimolar mixture of imidazole and MOPS produces an iron-loading curve that is intermediate between the imidazole and MOPS curves indicating that one buffer does not dominate the reaction mechanism. The UV-visible spectrum of the ferritin mineral has a higher absorbance from 250 to 450 nm when prepared in imidazole buffer than in MOPS buffer. These results suggest that different mineral phases form in ferritin by different loading mechanisms in imidazole and MOPS buffered reactions. Samples of 1500 Fe/ferritin were prepared in MOPS or imidazole buffer and were analyzed for crystallinity and using the electron diffraction capabilities of the electron microscope. The sample prepared in imidazole was significantly more crystalline than the sample prepared in MOPS. X-ray powder diffraction studies showed that small cores (~ 500 Fe/ferritin) prepared in MOPS or imidazole possess a 2-line ferrihydrite spectrum. As the core size increases the mineral phase begins to change from 2-line to 6-line ferrihydrite with the imidazole sample favoring the 6-line ferrihydrite phase. Taken together, these results suggest that the iron deposition mechanism in ferritin can be controlled by properties of the buffer with samples prepared in imidazole forming a larger, more ordered crystalline mineral than samples prepared in MOPS.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

The influence of reaction temperature on biomineralization of ferrihydrite cores in human H-ferritin

Ferritin is not only important for iron storage and detoxification in living organisms, but a multifunctional size-constrained nanoplatform for biomimetic nanoparticles. In order to tailor the biomimetic nanoparticles for future applications, it is essential to investigate the effects of external factors such as temperature on the particle size and structure of reconstituted cores in ferritin. In this study, we systematically investigated the mineral composition, crystallinity, and particle size of human H-ferritin (HuHF) reconstituted at four different temperatures (25, 30, 37, and 42°C) by integrated magnetic and transmission electron microscopy analyses. Our results showed that the particle size of reconstituted ferrihydrite cores (~5 nm) in HuHF was temperature-independent. However, the significant changes of the induced magnetization at 5 T field (M_5T) and remanent magnetization (M_r) at 5 K clearly showed that the crystallinity of reconstituted cores increased with increasing temperature, indicating that the reaction temperature deeply affects the structural order of reconstituted ferrihydrite cores rather than the particle size, and the reconstituted cores become more ordered at higher reaction temperatures. Our findings provide useful insights into biomineralization of ferritin under in vivo fever condition as well as in biomimetic synthesis of nanomaterials using ferritin. Furthermore, the rock magnetic methods should be very useful approaches for characterizing finite ferritin nanoparticles.     

Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus)

Ferritin is a conserved iron binding protein existing ubiquitously in prokaryotes and eukaryotes. In this study, the gene encoding a ferritin M subunit homologue (SoFer1) was cloned from red drum (Sciaenops ocellatus) and analyzed at expression and functional levels. The open reading frame of SoFer1 is 531 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE) preserved in many ferritins. The deduced amino acid sequence of SoFer1 possesses both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Expression of SoFer1 was tissue specific and responded positively to experimental challenges with Gram-positive and Gram-negative fish pathogens. Treatment of red drum liver cells with iron, copper, and oxidant significantly upregulated the expression of SoFer1 in time-dependent manners. To further examine the potential role of SoFer1 in antioxidation, red drum liver cells transfected transiently with SoFer1 were prepared. Compared to control cells, SoFer1 transfectants exhibited reduced production of reactive oxygen species following H₂O₂ challenge. Finally, to examine the iron binding potential of SoFer1, SoFer1 was expressed in and purified from Escherichia coli as a recombinant protein. Iron-chelating analysis showed that purified recombinant SoFer1 was capable of iron binding. Taken together, these results suggest that SoFer1 is likely to be a functional ferritin involved in iron sequestration, host immune defence against bacterial infection, and antioxidation.     

FERRITIN IN THE FUNGUS PHYCOMYCES

The iron-protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. It has a protein-to-iron ratio of 5, a sedimentation coefficient of 55S, a buoyant density in CsCl of 1.82 g/cm³, and the characteristic morphology of ferritin in the electron microscope. Apoferritin prepared from Phycomyces ferritin has a sedimentation coefficient of 18S and consists of subunits of molecular weight 25,000. In the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5–2.0 µ in diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin-sections. Ferritin is found in all developmental stages of Phycomyces but is concentrated in spores. The level of ferritin iron is regulated by the iron level in the growth medium, a 50-fold increase occurring on iron-supplemented medium.     

Nitrous Oxide Production in Sputum from Cystic Fibrosis Patients with Chronic Pseudomonas aeruginosa Lung Infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O₂ for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N₂O), an intermediate in the denitrification pathway. We measured N₂O and O₂ with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO₃ ⁻ and NO₂ ⁻ in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N₂O (range 1.4–157.9 µM N₂O). The concentration of N₂O in the sputum was higher below the oxygenated layers. In 4 samples the N₂O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO₃ ⁻ decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N₂O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.     

Controlled aggregation of ferritin to modulate MRI relaxivity

Ferritin is an iron storage protein expressed in varying concentrations in mammalian cells. The deposition of ferric iron in the core of ferritin makes it a magnetic resonance imaging contrast agent, and ferritin has recently been proposed as a gene expression reporter protein for magnetic resonance imaging. To date, ferritin has been overexpressed in vivo and has been coexpressed with transferrin receptor to increase iron loading in cells. However, ferritin has a relatively low T₂ relaxivity (R₂ ≈ 1 mM⁻¹s⁻¹) at typical magnetic field strengths and so requires high levels of expression to be detected. One way to modulate the transverse relaxivity of a superparamagnetic agent is to cause it to aggregate, thereby manipulating the magnetic field gradients through which water diffuses. In this work, it is demonstrated by computer simulation and in vitro that aggregation of ferritin can alter relaxivity. The effects of aggregate size and intraaggregate perturber spacing on R₂ are studied. Computer modeling indicates that the optimal spacing of the ferritin molecules in aggregate for increasing R₂ is 100-200 nm for a typical range of water diffusion rates. Chemical cross-linking of ferritin at 12 Å spacing led to a 70% increase in R₂ compared to uncross-linked ferritin controls. To modulate ferritin aggregation in a potentially biologically relevant manner, ferritin was attached to actin and polymerized in vitro. The polymerization of ferritin-F-actin caused a 20% increase in R₂ compared to unpolymerized ferritin-G-actin. The R₂-value was increased by another 10% by spacing the ferritin farther apart on the actin filaments. The modulation of ferritin aggregation by binding to cytoskeletal elements may be a useful strategy to make a functional reporter gene for magnetic resonance imaging.     

Ferritin—A general metal detoxicant

Binding of nonferrous metal ions to ferritin was compared to that of the phosphate-free or phosphate containing synthetic iron cores. The Scatchard plots for the synthetic cores reveal a high affinity site for Cd, Zn, Be, and Al, with K_D in the range 10^?5–10^?7 M. Preloading the cores with phosphate increased the number of metal ions bound without altering the K_D. The metal ions with smaller ionic radii (Be, Al) were bound in larger numbers than those with larger ionic radii (Cd, Zn). Ferritin isolated from soybean (Glycina max), horse spleen, and rat liver bound the metal ions in amounts larger than predicted from their iron core. Whereas the iron cores and their nonferrous metal ion complexes were insoluble, those in the protein shell remained in solution. Thus apoferritin precipitated with lower concentrations of aluminum than did holoferritin. Also, Al bound to apoferritin reduced the rate of iron loading into the protein.     

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, ⁵⁵Fe uptake assays, and LC–MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.     

Purification and Properties of Ferritin from the Last Instar Larval Hemolymph of Protaetia brevitarsis (Coleoptera)

Ferritin, an iron storage protein, has been purified from the last larval hemolymph of Protaetia brevitarsis (coleoptera) by KBr density gradient ultracentrifugation and resource Q (anion exchange chromatography) using fast performance liquid chromatography (FPLC) system. The iron content of ferritin was determined by atomic emission spectroscopy and FereneS stain. Ferritin of P. brevitarsis is shown to have two different subunits presented on a SDSPAGE in normal (N) and ironinjected (I) hemolymph. SDS PAGE revealed that the ferritin consists of two major polypeptides of 27 and 28 kDa in normal hemolymph. Interestingly, however, 30 kDa subunit was substituted for 28 kDa when iron was injected into the hemolymph. Apporximate isoelectric points of 27 kDa, 28 kDa, and 30 kDa ferritin subunits were 6.7, 6.75, and 6.8, respectively. Ferritin of P. brevitarsis was detected by FereneS stain and confirmed by Western blotting using its polyclonal antibody. Other characteristics such as amino acid composition and Nterminal amino acid sequence were investigated. Amino acid composition of ferritin (N and I) was rich in alanine, glycine, glutamine or glutamic acid and serine, but poor in histidine, arginine, methionine and phenylalanine.     

Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2)

Iron is essential to plants. However, when free and in excess, iron can catalyze the formation of oxygen free radicals. Ferritin, a protein capable of storing up to 4500 atoms of iron, can act as an iron buffer inside plant cells. Using a strategy based in amplicon size difference, we were able to analyze the expression profile of the two rice ferritin genes (OsFER1 and OsFER2). Both genes are expressed, although with different regulation and organ distribution. Exposure to copper, Paraquat, SNP and excess iron led to accumulation of ferritin mRNA, remarkably of OsFER2. The iron-induced expression was abolished by treatment with GSH, indicating that the induction observed is dependent of an oxidative step. OsFER2 mRNA levels in rice flag leaves and panicles at different reproductive stages were higher than OsFER1 mRNA levels. No ferritin mRNA was detected in rice seeds. However, imbibition under light led to ferritin expression, which was abolished when seeds were kept in the dark, suggesting a light-regulated induction. Ferritin mRNA accumulation was seen in the dark only when seeds were germinated in the presence of externally supplied iron. We suggest that the primary role of rice ferritins is related to defense against iron-mediated oxidative stress.     

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases?

Background

The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer…) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form.

Scope

Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients.

Major conclusions

Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric–ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity.

General significance

The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.