Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

Ferritin microheterogeneity, subunit composition, functional, and physiological implications.

Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.

Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design.

Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co-assemble in different H and L ratios to form 24-mer shell-like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure-function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L-rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H-ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron-induced protein thermal destabilization effect (ΔTm up to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo- and hetero-polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L-rich ferritins may have great potential for biotechnological applications.

Ferritin nanocages as efficient nanocarriers and promising platforms for COVID-19 and other vaccines development.

BACKGROUND: The development of safe and effective vaccines against SARS-CoV-2 and other viruses with high antigenic drift is of crucial importance to public health. Ferritin is a well characterized and ubiquitous iron storage protein that has emerged not only as a useful nanoreactor and nanocarrier, but more recently as an efficient platform for vaccine development. SCOPE OF REVIEW: This review discusses ferritin structure-function properties, self-assembly, and novel bioengineering strategies such as interior cavity and exterior surface modifications for cargo encapsulation and delivery. It also discusses the use of ferritin as a scaffold for biomedical applications, especially for vaccine development against influenza, Epstein-Barr, HIV, hepatitis-C, Lyme disease, and respiratory viruses such as SARS-CoV-2. The use of ferritin for the synthesis of mosaic vaccines to deliver a cocktail of antigens that elicit broad immune protection against different viral variants is also explored. MAJOR CONCLUSIONS: The remarkable stability, biocompatibility, surface functionalization, and self-assembly properties of ferritin nanoparticles make them very attractive platforms for a wide range of biomedical applications, including the development of vaccines. Strong immune responses have been observed in pre-clinical studies against a wide range of pathogens and have led to the exploration of ferritin nanoparticles-based vaccines in multiple phase I clinical trials. GENERAL SIGNIFICANCE: The broad protective antibody response of ferritin nanoparticles-based vaccines demonstrates the usefulness of ferritin as a highly promising and effective approaches for vaccine development.

Effect of Phosphate and Ferritin Subunit Composition on the Kinetics, Structure, and Reactivity of the Iron Core in Human Homo- and Heteropolymer Ferritins.

Ferritins are highly conserved supramolecular protein nanostructures that play a key role in iron homeostasis. Thousands of iron atoms can be stored inside their hollow cavity as a hydrated ferric oxyhydroxide mineral. Although phosphate associates with the ferritin iron nanoparticles, the effect of physiological concentrations on the kinetics, structure, and reactivity of ferritin iron cores has not yet been explored. Here, the iron loading and mobilization kinetics were studied in the presence of 1-10 mM phosphate using homopolymer and heteropolymer ferritins having different H to L subunit ratios. In the absence of ferritin, phosphate enhances the rate of ferrous ion oxidation and forms large and soluble polymeric Fe(III)-phosphate species. In the presence of phosphate, Fe(II) oxidation and core formation in ferritin is significantly accelerated with oxidation rates several-fold higher than with phosphate alone. High-angle annular dark-field scanning transmission electron microscopy measurements revealed a strong phosphate effect on both the size and morphology of the iron mineral in H-rich (but not L-rich) ferritins. While iron nanoparticles in L-rich ferritins have spherical shape in the absence and presence of phosphate, iron nanoparticles in H-rich ferritins change from irregular shapes in the absence of phosphate to spherical particles in the presence of phosphate with larger size distribution and smaller particle size. In the presence of phosphate, the kinetics of iron-reductive mobilization from ferritin releases twice as much iron than in its absence. Altogether, our results demonstrate an important role for phosphate, and the ferritin H and L subunit composition toward the kinetics of iron oxidation and removal from ferritin, as well as the structure and reactivity of the iron mineral, and may have an important implication on ferritin iron management in vivo.

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Micromagnetic and morphological characterization of heteropolymer human ferritin cores.

The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), electron energy-loss spectroscopy (EELS), and 57Fe Mössbauer spectroscopy were employed to ascertain (1) the microstructural, electronic, and micromagnetic properties of the nanosized iron cores, and (2) the effect of the H and L ferritin subunit ratios on these properties. Mössbauer spectroscopic signatures indicate that all iron within the core is in the high spin ferric state. Variable temperature Mössbauer spectroscopy for H-rich (H21/L3) and L-rich (H2/L22) ferritins reconstituted at 1000 57Fe/protein indicates superparamagnetic behavior with blocking temperatures of 19 K and 28 K, while HAADF-STEM measurements give average core diameters of (3.7 ± 0.6) nm and (5.9 ± 1.0) nm, respectively. Most significantly, H-rich proteins reveal elongated, dumbbell, and crescent-shaped cores, while L-rich proteins present spherical cores, pointing to a correlation between core shape and protein shell composition. Assuming an attempt time for spin reversal of τ 0 = 10-11 s, the Néel-Brown formula for spin-relaxation time predicts effective magnetic anisotropy energy densities of 6.83 × 104 J m-3 and 2.75 × 104 J m-3 for H-rich and L-rich proteins, respectively, due to differences in surface and shape contributions to magnetic anisotropy in the two heteropolymers. The observed differences in shape, size, and effective magnetic anisotropies of the derived biomineral cores are discussed in terms of the iron nucleation sites within the interior surface of the heteropolymer shells for H-rich and L-rich proteins. Overall, our results imply that site-directed nucleation and core growth within the protein cavity play a determinant role in the resulting core morphology. Our findings have relevance to iron biomineralization processes in nature and the growth of designer's magnetic nanoparticles within recombinant apoferritin nano-templates for nanotechnology.

A Novel Approach for the Synthesis of Human Heteropolymer Ferritins of Different H to L Subunit Ratios.

Mammalian ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types, named H and L, that co-assemble in different proportions. Despite their discovery more than 8 decades ago, recombinant human heteropolymer ferritins have never been synthesized, owing to the lack of a good expression system. Here, we describe for the first time a unique approach that uses a novel plasmid design that enables the synthesis of these complex ferritin nanostructures. Our study reveals an original system that can be easily tuned by altering the concentrations of two inducers, allowing the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between (isoferritins). The H to L subunit composition of purified ferritin heteropolymers was analyzed by SDS-PAGE and capillary gel electrophoresis, and their iron handling properties characterized by light absorption spectroscopy. Our novel approach allows future investigations of the structural and functional differences of isoferritin populations, which remain largely obscure. This is particularly exciting since a change in the ferritin H- to L-subunit ratio could potentially lead to new iron core morphologies for various applications in bio-nanotechnologies.

Complexation of ferrous ions by ferrozine, 2,2'-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems.

Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe2+) and oxidized (Fe3+) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV-vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2'-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe2+-chelator complexes. Our results show that the kinetics of the formation of the {Fe2+-(ferrozine)3} complex, but not the{Fe2+-(bipyridine)3} or the {Fe(II)-(phenanthroline)3} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 M-1 cm-1 for ferrozine at 562 nm, 9950 ± 100 M-1 cm-1 for 2,2'-bipyridine at 522 nm, and 12,450 ± 370 M-1 cm-1 for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.

A rapid, efficient and sensitive plate assay for detection and screening of l-asparaginase-producing microorganisms.

l-Asparaginase-producing microbes are conventionally screened on phenol red l-asparagine-containing plates. However, sometimes the contrast of the zone obtained (between yellow and pink) is not very sharp and distinct. In the present investigation, an improved method for screening of the microorganisms producing extracellular l-asparaginase is reported wherein bromothymol blue (BTB) is incorporated as pH indicator in l-asparagine-containing medium instead of phenol red. Plates containing BTB at acidic pH are yellow and turn dark blue at alkaline pH. Thus, a dense dark blue zone is formed around microbial colonies producing l-asparaginase, differentiating between enzyme producers and non-producers. The present method is more sensitive and accurate than the conventional method for screening of both fungi and bacteria producing extracellular l-asparaginase. Furthermore, BTB gives a transient green colour at neutral pH (7.0) and dark blue colour at higher pH 8.0-9.0, indicating the potency of the microorganism for l-asparaginase production.