RESUMEN
Ferritin is a 24-mer protein nanocage that stores iron and regulates intracellular iron homeostasis. The nuclear receptor coactivator-4 (NCOA4) binds specifically to ferritin H subunits and facilitates the autophagic trafficking of ferritin to the lysosome for degradation and iron release. Using isothermal titration calorimetry (ITC), we studied the thermodynamics of the interactions between ferritin and the soluble fragment of NCOA4 (residues 383-522), focusing on the effects of the recently identified FeS cluster bound to NCOA4, ferritin subunit composition, and ferritiniron loading. Our findings show that in the presence of the FeS cluster, the binding is driven by a more favorable enthalpy change and a decrease in entropy change, indicating a key role for the FeS cluster in the structural organization and stability of the complex. The ferritin iron core further enhances this association, increasing binding enthalpy and stabilizing the NCOA4-ferritin complex. The ferritin subunit composition primarily affects binding stoichiometry of the reaction based on the number of H subunits in the ferritin H/L oligomer. Our results demonstrate that both the FeS cluster and the ferritin iron core significantly affect the binding thermodynamics of the NCOA4-ferritin interactions, suggesting regulatory roles for the FeS cluster and ferritin iron content in ferritinophagy.
RESUMEN
We report a simple and highly sensitive colorimetric method for the detection and quantification of proteins, based on the aggregation of ascorbic acid (AA) capped gold nanoparticles (AuNPs) by proteins. The interactions between our AuNPs and nine different proteins of various sizes and shapes (cytochrome C (12 kDa), lysozyme (14.3 kDa), myoglobin (17 kDa), human serum albumin (66 kDa), bovine serum albumin (66.4 kDa), human transferrin (80 kDa), aldolase (160 kDa), catalase (240 kDa), and human H-ferritin (500 kDa)) generated similar AuNPs-protein absorption spectra in a concentration-dependent manner in the range of 1-15 nM. Upon the addition of a protein, the UV-visible spectra of AuNPs-protein conjugates shifted from 524 nm for the AuNps alone to longer wavelength (600-750 nm) due to the presence of one of these proteins. This bathochromic shift is accompanied by a color change from a cherry red, to dark purple, and then light grey or colorless if excess protein has been added, indicating the formation of AuNPs-protein conjugates followed by protein-induced aggregation of the AuNPs. High-resolution transmission electron microscopy images revealed uniformly distributed spherical nanoparticles with an average size of 27.5 ± 15.2 nm, increasing in size to 39.6 ± 12.9 nm upon the addition of a protein, indicating the formation of AuNPs-protein conjugates in solution. A general mechanism for the protein-induced aggregation of our AuNPs is proposed. The consistent behavior observed with the nine proteins tested in our study suggests that our assay can be universally applied for the quantification of pure proteins in a solution, regardless of size, shape, or molecular weight.
Asunto(s)
Ácido Ascórbico , Colorimetría , Oro , Nanopartículas del Metal , Proteínas , Oro/química , Ácido Ascórbico/química , Colorimetría/métodos , Nanopartículas del Metal/química , Humanos , Proteínas/química , Proteínas/análisis , Animales , Bovinos , Espectrofotometría Ultravioleta/métodosRESUMEN
Despite ferritin's critical role in regulating cellular and systemic iron levels, our understanding of the structure and assembly mechanism of isoferritins, discovered over eight decades ago, remains limited. Unveiling how the composition and molecular architecture of hetero-oligomeric ferritins confer distinct functionality to isoferritins is essential to understanding how the structural intricacies of H and L subunits influence their interactions with cellular machinery. In this study, ferritin heteropolymers with specific H to L subunit ratios were synthesized using a uniquely engineered plasmid design, followed by high-resolution cryo-electron microscopy analysis and deep learning-based amino acid modeling. Our structural examination revealed unique architectural features during the self-assembly mechanism of heteropolymer ferritins and demonstrated a significant preference for H-L heterodimer formation over H-H or L-L homodimers. Unexpectedly, while dimers seem essential building blocks in the protein self-assembly process, the overall mechanism of ferritin self-assembly is observed to proceed randomly through diverse pathways. The physiological significance of these findings is discussed including how ferritin microheterogeneity could represent a tissue-specific adaptation process that imparts distinctive tissue-specific functions to isoferritins.
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Ferritinas , Multimerización de Proteína , Humanos , Ferritinas/química , Ferritinas/metabolismo , Ferritinas/genética , Modelos Moleculares , Microscopía por CrioelectrónRESUMEN
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.
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Microscopía por Crioelectrón , Ferritinas , Coactivadores de Receptor Nuclear , Ferritinas/metabolismo , Ferritinas/química , Ferritinas/genética , Humanos , Coactivadores de Receptor Nuclear/metabolismo , Coactivadores de Receptor Nuclear/química , Coactivadores de Receptor Nuclear/genética , Unión Proteica , Sitios de Unión , Hierro/metabolismo , Autofagia , Modelos Moleculares , Células HEK293 , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Proteolisis , MutaciónRESUMEN
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.
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Ferritinas , Hierro , Animales , Ferritinas/metabolismo , Focalización Isoeléctrica , Electroforesis en Gel de Poliacrilamida , Hierro/metabolismo , Punto IsoeléctricoRESUMEN
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.
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Ferritinas , Hierro , Animales , Humanos , Ferritinas/genética , Ferritinas/química , Hierro/metabolismo , Polímeros/metabolismo , Plásmidos/genética , Proteínas Recombinantes/química , MamíferosRESUMEN
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.
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COVID-19 , Vacunas contra la Influenza , Humanos , Ferritinas , COVID-19/prevención & control , Vacunas contra la COVID-19 , SARS-CoV-2 , Desarrollo de VacunasRESUMEN
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.
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Ferritinas , Hierro , Apoferritinas/metabolismo , Compuestos Férricos/química , Ferritinas/química , Compuestos Ferrosos/metabolismo , Humanos , Hierro/química , Cinética , Fosfatos/metabolismoRESUMEN
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.
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Ferritinas , Hierro , Transporte de Electrón , Ferritinas/metabolismo , Hierro/metabolismo , Cinética , Riboflavina/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
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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.
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Apoferritinas/metabolismo , Plásmidos/genética , Ingeniería de Proteínas/métodos , Apoferritinas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Humanos , Hierro/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Mag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. METHODS: Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 µM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. RESULTS: The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105µM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) µM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10). CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. GENERAL SIGNIFICANCE: Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.
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Calcio/metabolismo , Colorantes Fluorescentes/metabolismo , Fura-2/análogos & derivados , Músculo Esquelético/metabolismo , Animales , Colorantes Fluorescentes/química , Fura-2/química , Fura-2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/química , TermodinámicaRESUMEN
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.
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2,2'-Dipiridil/química , Quelantes/química , Complejos de Coordinación/química , Ferrozina/química , Hierro/química , Fenantrolinas/química , Concentración de Iones de Hidrógeno , Cinética , LigandosRESUMEN
BACKGROUND: The mechanism of iron oxidation and core formation in homopolymeric H-type ferritins has been extensively studied in-vitro, so has the reductive mobilization of iron from the inorganic iron(III) core. However, neither process is well-understood in-vivo despite recent scientific advances. SCOPE OF REVIEW: Here, we provide a summary of our current understanding of iron mineralization and iron core dissolution in homopolymeric H-type ferritins and highlight areas of interest and further studies that could answer some of the outstanding questions of iron metabolism. MAJOR CONCLUSIONS: The overall iron oxidation mechanism in homopolymeric H-type ferritins from vertebrates (i.e. human H and frog M ferritins) is similar, despite nuances in the individual oxidation steps due to differences in the iron ligand environments inside the three fold channels, and at the dinuclear ferroxidase centers. Ferrous cations enter the protein shell through hydrophilic channels, followed by their rapid oxidization at diiron centers. Hydrogen peroxide produced during iron oxidation can react with additional iron(II) at ferroxidase centers, or at separate sites, or possibly on the surface of the mineral core. In-vitro ferritin iron mobilization can be achieved using a variety of reducing agents, but in-vivo iron retrieval may occur through a variety of processes, including proteolytic degradation, auxiliary iron mobilization mechanisms involving physiological reducing agents, and/or oxidoreductases. GENERAL SIGNIFICANCE: This review provides important insights into the mechanisms of iron oxidation and mobilization in homopolymeric H-type ferritins, and different strategies in maintaining iron homeostasis.
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Apoferritinas/metabolismo , Hierro/metabolismo , Animales , Apoferritinas/química , Transporte Biológico , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Humanos , Modelos Moleculares , Oxidación-Reducción , ProteolisisRESUMEN
Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of â¼8 NCOA4 molecules per ferritin shell, and Kd values of â¼0.4 and â¼2 µM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.
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Apoferritinas/metabolismo , Ferritinas/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Oxidorreductasas/metabolismo , Apoferritinas/química , Sitios de Unión , Ferritinas/química , Humanos , Cinética , Coactivadores de Receptor Nuclear/química , Oxidorreductasas/química , Unión Proteica , Mapas de Interacción de Proteínas , TermodinámicaRESUMEN
Fenretinide is a synthetic retinoid pharmaceutical linked to ceramide build-up in vivo. Saposin D is an intralysosomal protein necessary for ceramide binding/degradation. We show, via electronic absorption spectroscopy, fluorescence spectroscopy, and ceramide hydrolysis assays, that fenretinide is bound by saposin D {K a = (1.45 ± 0.49) × 105 M-1}, and affects ceramide solubilization/degradation.
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In mammals, the iron storage and detoxification protein ferritin is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light). The two subunits co-assemble in various ratios, with a tissue specific distribution, to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunits possess ferroxidase centers that catalyze the rapid oxidation of ferrous ions, whereas the L-subunit does not have such centers and is believed to play an important role in electron transfer reactions that occur during the uptake and release of iron. Pathogenic mutations on the L-chain lead to neuroferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin inclusion bodies and iron in the central nervous system. Here, we have characterized the thermal stability, iron loading capacity, iron uptake, and iron release properties of ferritin heteropolymers carrying the three pathogenic L-ferritin mutants (L154fs, L167fs, and L148fs, which for simplicity we named Ln1, Ln2 and Ln3, respectively), and a non-pathogenic variant (L135P) bearing a single substitution on the 3-fold axes of L-subunits. The UV-Vis data show a similar iron loading capacity (ranging between 1800 to 2400 Fe(iii)/shell) for all ferritin samples examined in this study, with Ln2 holding the least amount of iron (i.e. 1800 Fe(iii)/shell). The three pathogenic L-ferritin mutants revealed higher rates of iron oxidation and iron release, suggesting that a few mutated L-chains on the heteropolymer have a significant effect on iron permeability through the ferritin shell. DSC thermograms showed a strong destabilization effect, the severity of which depends on the location of the frameshift mutations (i.e. wt heteropolymer ferritin â homopolymer H-chain > L135P > Ln2 > Ln1 > Ln3). Variant L135P had only minor effects on the protein functionality and stability, suggesting that local melting of the 3-fold axes in this variant may not be responsible for neuroferritinopathy-like disorders. The data support the hypothesis that hereditary neuroferritinopathies are due to alterations of ferritin functionality and lower physical stability which correlate with the frameshifts introduced at the C-terminal sequence and explain the dominant transmission of the disorder.
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Apoferritinas/genética , Apoferritinas/metabolismo , Trastornos del Metabolismo del Hierro/genética , Hierro/metabolismo , Distrofias Neuroaxonales/genética , Apoferritinas/química , Humanos , Trastornos del Metabolismo del Hierro/metabolismo , Modelos Moleculares , Distrofias Neuroaxonales/metabolismo , Oxidación-Reducción , Mutación Puntual , Estabilidad Proteica , Desplegamiento ProteicoRESUMEN
The excessively high and inconsistent literature values for Km,Fe and Km,O2 prompted us to examine the iron oxidation kinetics in ferritin, the major iron storage protein in mammals, and to determine whether a traditional Michaelis-Menten enzymatic behavior is obeyed. The kinetics of Fe(ii) oxidation and mineralization catalyzed by three different types of ferritins (recombinant human homopolymer 24H, HuHF, human heteropolymer â¼21H:3L, HL, and horse spleen heteropolymer â¼3.3H:20.7L, HosF) were therefore studied under physiologically relevant O2 concentrations, but also in the presence of excess Fe(ii) and O2 concentrations. The observed iron oxidation kinetics exhibited two distinct phases (phase I and phase II), neither of which obeyed Michaelis-Menten kinetics. While phase I was very rapid and corresponded to the oxidation of approximately 2 Fe(ii) ions per H-subunit, phase II was much slower and varied linearly with the concentration of iron(ii) cations in solution, independent of the size of the iron core. Under low oxygen concentration close to physiological, the iron uptake kinetics revealed a Michaelis-Menten behavior with Km,O2 values in the low µM range (i.e. â¼1-2 µM range). Our experimental Km,O2 values are significantly lower than typical cellular oxygen concentration, indicating that iron oxidation and mineralization in ferritin should not be affected by the oxygenation level of cells, and should proceed even under hypoxic events. A kinetic model is proposed in which the inhibition of the protein's activity is caused by bound iron(iii) cations at the ferroxidase center, with the rate limiting step corresponding to an exchange or a displacement reaction between incoming Fe(ii) cations and bound Fe(iii) cations.
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Ferritinas/metabolismo , Hierro/metabolismo , Oxígeno/metabolismo , Animales , Caballos , Humanos , Cinética , Oxidación-Reducción , Proteínas Recombinantes/metabolismo , Bazo/metabolismoRESUMEN
Ferritins are highly conserved supramolecular protein nanostructures composed of two different subunit types, H (heavy) and L (light). The two subunits co-assemble into a 24-subunit heteropolymer, with tissue specific distributions, to form shell-like protein structures within which thousands of iron atoms are stored as a soluble inorganic ferric iron core. In-vitro (or in cell free systems), the mechanisms of iron(II) oxidation and formation of the mineral core have been extensively investigated, although it is still unclear how iron is loaded into the protein in-vivo. In contrast, there is a wide spread belief that the major pathway of iron mobilization from ferritin involves a lysosomal proteolytic degradation of ferritin, and the dissolution of the iron mineral core. However, it is still unclear whether other auxiliary iron mobilization mechanisms, involving physiological reducing agents and/or cellular reductases, contribute to the release of iron from ferritin. In vitro iron mobilization from ferritin can be achieved using different reducing agents, capable of easily reducing the ferritin iron core, to produce soluble ferrous ions that are subsequently chelated by strong iron(II)-chelating agents. Here, we review our current understanding of iron mobilization from ferritin by various reducing agents, and report on recent results from our laboratory, in support of a mechanism that involves a one-electron transfer through the protein shell to the iron mineral core. The physiological significance of the iron reductive mobilization from ferritin by the non-enzymatic FMN/NAD(P)H system is also discussed.
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Saposin B (SapB) is a human lysosomal protein, critical for the degradation of O-sulfogalactosylceramide (sulfatide). SapB binds sulfatide and presents it to the active site of the enzyme arylsulfatase A. Deficiency of SapB leads to fatal activator-deficient metachromatic leukodystrophy. Given the conformational flexibility and the large hydrophobic "pocket" produced upon (physiologically relevant) homodimerization, SapB may have broader substrate diversity than originally thought. Herein, we present evidence using fluorescence spectroscopy and computational docking studies that SapB binds a wide variety of ligands at KD values varying from micromolar to nanomolar, with entropy being the primary driving force. We further demonstrate, for the first time, that SapB has two binding sites that can sequentially (and in a preferred order) accommodate up to two ligands at once.