scholarly journals Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres

1996 ◽  
Vol 314 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Paolo SANTAMBROGIO ◽  
Sonia LEVI ◽  
Anna COZZI ◽  
Barbara CORSI ◽  
Paolo AROSIO
Keyword(s):  
Storage Proteins ◽  
Iron Storage ◽  
Cellular Iron ◽  
Iron Incorporation ◽  
Ferroxidase Activity ◽  
Iron Storage Proteins ◽  
The Difference ◽  
Induced Aggregation

Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.

scholarly journals A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

2012 ◽  
Vol 80 (10) ◽  
pp. 3650-3659 ◽  
Author(s):  
Ruchi Pandey ◽  
G. Marcela Rodriguez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Iron Homeostasis ◽  
Storage Proteins ◽  
Iron Acquisition ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Content Type ◽  
Iron Storage Proteins

ABSTRACTIron is an essential, elusive, and potentially toxic nutrient for most pathogens, includingMycobacterium tuberculosis. Due to the poor solubility of ferric iron under aerobic conditions, free iron is not found in the host.M. tuberculosisrequires specialized iron acquisition systems to replicate and cause disease. It also depends on a strict control of iron metabolism and intracellular iron levels to prevent iron-mediated toxicity. Under conditions of iron sufficiency,M. tuberculosisrepresses iron acquisition and induces iron storage, suggesting an important role for iron storage proteins in iron homeostasis.M. tuberculosissynthesizes two iron storage proteins, a ferritin (BfrB) and a bacterioferritin (BfrA). The individual contributions of these proteins to the adaptive response ofM. tuberculosisto changes in iron availability are not clear. By generating individual knockout strains ofbfrAandbfrB, the contribution of each one of these proteins to the maintenance of iron homeostasis was determined. The effect of altered iron homeostasis, resulting from impaired iron storage, on the resistance ofM. tuberculosistoin vitroandin vivostresses was examined. The results show that ferritin is required to maintain iron homeostasis, whereas bacterioferritin seems to be dispensable for this function.M. tuberculosislacking ferritin suffers from iron-mediated toxicity, is unable to persist in mice, and, most importantly, is highly susceptible to killing by antibiotics, showing that endogenous oxidative stress can enhance the antibiotic killing of this important pathogen. These results are relevant for the design of new therapeutic strategies againstM. tuberculosis.

scholarly journals Dps and DpsL Mediate SurvivalIn VitroandIn Vivoduring the Prolonged Oxidative Stress Response in Bacteroides fragilis

2015 ◽  
Vol 197 (20) ◽  
pp. 3329-3338 ◽  
Author(s):  
Michael I. Betteken ◽  
Edson R. Rocha ◽  
C. Jeffrey Smith
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Peritoneal Cavity ◽  
Bacteroides Fragilis ◽  
Oxidative Stress Response ◽  
Iron Storage ◽  
Content Type ◽  
Iron Storage Proteins

ABSTRACTBacteroides fragilisis a Gram-negative anaerobe and member of the human intestinal tract microbiome, where it plays many beneficial roles. However, translocation of the organism to the peritoneal cavity can lead to peritonitis, intra-abdominal abscess formation, bacteremia, and sepsis. During translocation,B. fragilisis exposed to increased oxidative stress from the oxygenated tissues of the peritoneal cavity and the immune response. In order to survive,B. fragilismounts a robust oxidative stress response consisting of an acute and a prolonged oxidative stress (POST) response. This report demonstrates that the ability to induce high levels of resistance totert-butyl hydroperoxide (tBOOH) after extended exposure to air can be linked to the POST response. Disk diffusion assays comparing the wild type to a Δdpsmutant and a ΔdpsΔbfrmutant showed greater sensitivity of the mutants to tBOOH after exposure to air, suggesting that Dps and DpsL play a role in the resistance phenotype. Complementation studies withdpsorbfr(encoding DpsL) restored tBOOH resistance, suggesting a role for both of these ferritin-family proteins in the response. Additionally, cultures treated with the iron chelator dipyridyl were not killed by tBOOH, indicating Dps and DpsL function by sequestering iron to prevent cellular damage. Anin vivoanimal model showed that the ΔdpsΔbfrmutant was attenuated, indicating that management of iron is important for survival within the abscess. Together, these data demonstrate a role for Dps and DpsL in the POST response which mediates survivalin vitroandin vivo.IMPORTANCEB. fragilisis the anaerobe most frequently isolated from extraintestinal opportunistic infections, but there is a paucity of information about the factors that allow this organism to survive outside its normal intestinal environment. This report demonstrates that the iron storage proteins Dps and DpsL protect against oxidative stress and that they contribute to survival bothin vitroandin vivo. Additionally, this work demonstrates an important role for the POST response inB. fragilissurvival and provides insight into the complex regulation of this response.

The importance of eukaryotic ferritins in iron handling and cytoprotection

2015 ◽  
Vol 472 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Paolo Arosio ◽  
Fernando Carmona ◽  
Raffaella Gozzelino ◽  
Federica Maccarinelli ◽  
Maura Poli
Keyword(s):  
Immune Response ◽  
Innate Immunity ◽  
Animal Models ◽  
Storage Proteins ◽  
Eukaryotic Cells ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Incorporation ◽  
Iron Storage Proteins ◽  
Response Activation

Ferritins, the main intracellular iron storage proteins, have been studied for over 60 years, mainly focusing on the mammalian ones. This allowed the elucidation of the structure of these proteins and the mechanisms regulating their iron incorporation and mineralization. However, ferritin is present in most, although not all, eukaryotic cells, comprising monocellular and multicellular invertebrates and vertebrates. The aim of this review is to provide an update on the general properties of ferritins that are common to various eukaryotic phyla (except plants), and to give an overview on the structure, function and regulation of ferritins. An update on the animal models that were used to characterize H, L and mitochondrial ferritins is also provided. The data show that ferritin structure is highly conserved among different phyla. It exerts an important cytoprotective function against oxidative damage and plays a role in innate immunity, where it also contributes to prevent parenchymal tissue from the cytotoxicity of pro-inflammatory agonists released by the activation of the immune response activation. Less clear are the properties of the secretory ferritins expressed by insects and molluscs, which may be important for understanding the role played by serum ferritin in mammals.

scholarly journals Large protein organelles form a new iron sequestration system with high storage capacity

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Tobias W Giessen ◽  
Benjamin J Orlando ◽  
Andrew A Verdegaal ◽  
Melissa G Chambers ◽  
Jules Gardener ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Storage Capacity ◽  
Storage Proteins ◽  
Storage System ◽  
Redox Balance ◽  
Iron Storage ◽  
X Ray Crystallography ◽  
Cellular Iron ◽  
Iron Storage Proteins ◽  
Assembly Principles

Iron storage proteins are essential for cellular iron homeostasis and redox balance. Ferritin proteins are the major storage units for bioavailable forms of iron. Some organisms lack ferritins, and it is not known how they store iron. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo and show interactions underlying cargo-shell co-assembly. This compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. Our results reveal a new approach for survival in diverse habitats with limited or fluctuating iron availability via an iron storage system able to store 10 to 20 times more iron than ferritin.

scholarly journals Transient overexpression of human H- and L-ferritin chains in COS cells

1998 ◽  
Vol 330 (1) ◽  
pp. 315-320 ◽  
Author(s):  
Barbara CORSI ◽  
Federica PERRONE ◽  
Monique BOURGEOIS ◽  
Carole BEAUMONT ◽  
C. Maria PANZERI ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Ferritin Concentration ◽  
Transfected Cells ◽  
Cos Cells ◽  
Cellular Models ◽  
Cellular Iron ◽  
Iron Incorporation

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Reliability of a Single β-Thromboglobulin Measurement in a Diabetic Population : Importance of PGE1 in Anticoagulant Mixture

1987 ◽  
Vol 57 (02) ◽  
pp. 201-204 ◽  
Author(s):  
P Y Scarabin ◽  
L Strain ◽  
C A Ludlam ◽  
J Jones ◽  
E M Kohner
Keyword(s):  
In Vitro Release ◽  
Mean Value ◽  
Reliable Estimate ◽  
Diabetic Patients ◽  
Single Measurement ◽  
Diabetic Population ◽  
The Difference ◽  
Vitro Release

SummaryDuring the collection of samples for plasma β-thromboglobulin (β-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single β-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE!. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p <0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artifically high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGEi decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.

scholarly journals Distinct Effects of Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus Cell Wall Component-Induced Inflammation on the Iron Metabolism of THP-1 Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1497
Author(s):  
Edina Pandur ◽  
Kitti Tamási ◽  
Ramóna Pap ◽  
Gergely Jánosa ◽  
Katalin Sipos
Keyword(s):  
Cell Wall ◽  
Inflammatory Cytokines ◽  
Iron Metabolism ◽  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Storage Proteins ◽  
Iron Storage ◽  
E Coli ◽  
Iron Storage Proteins ◽  
Pro Inflammatory Cytokines

Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.

scholarly journals Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation

1999 ◽  
Vol 338 (3) ◽  
pp. 615-618 ◽  
Author(s):  
Xiaoke YANG ◽  
N. Dennis CHASTEEN
Keyword(s):  
Ph Control ◽  
Alternative Mechanism ◽  
Experimental Conditions ◽  
Iron Storage ◽  
Ferroxidase Activity ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Ph Buffer ◽  
Horse Spleen Ferritin

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.

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