Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin.

The cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX) is attributed to reactions between iron and reactive oxygen species (ROS) that lead to oxidative damage. We found that DOX forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and the expression of stress-responsive genes such as catalase or aldose reductase. DOX also increased ferritin levels in these cells, particularly the H subunit. A considerable increase in ferritin mRNA levels showed that DOX acted at transcriptional level, but an additional potential mechanism was identified as the down-regulation of iron regulatory protein-2, post-transcriptional inhibitor of ferritin synthesis. Pretreatment with DOX protected H9c2 cells against the damage induced by subsequent exposure to ferric ammonium citrate, and experiments with 55Fe revealed that the protection was due to the deposition of iron in ferritin. Cytoprotection was also observed when DOX was replaced by glucose/glucose oxidase, a source of H2O2, thus suggesting that DOX increases ferritin synthesis through the action of ROS. This concept was supported by three more lines of evidence. (i) DOX-induced ferritin synthesis was blocked by N-acetylcysteine, a scavenger of ROS. (ii) Mitoxantrone, a ROS-forming analogue, similarly induced ferritin expression and protected the cells against iron toxicity. (iii) 5-Iminodaunorubicin, an analogue lacking ROS-forming activity, did not induce ferritin synthesis or protect the cells against iron toxicity. These results characterize a paradoxically beneficial link between anthracycline-derived ROS, increased ferritin synthesis, and resistance to iron-mediated damage. The role of iron and ROS in anthracycline-induced cardiotoxicity may, therefore, be more complex than previously believed

Wearable device for swim assessment: a new ecologic approach for communication and analysis

This paper describes a wearable system for the assessment of swim performance focusing on the description of the system and especially on the novel method for data transmission in water, and the algorithm used for extracting some parameters for the qualitative assessment of the performance. We placed a 3-axes Inertial Measurement Unit (IMU) on the athlete's back in a specific swimsuit, which also allows for recording 1-lead ECG. The system and the algorithm have been tested on 13 trials with 10 subjects comparing the results with data extracted from video recording. The system demonstrated able and reliable to measure time and kinematic parameters of swimming

Role of iron and ferritin in TNFα-induced apoptosis in HeLa cells

AbstractWe found that tumor necrosis factor α (TNFα)-induced apoptosis in HeLa cells was accompanied by a ∼2-fold increase in H- and L-ferritin and a decrease in transferrin receptor, two indices of increased iron availability. Iron supplementation and overexpression of H-ferritin or its mutant with an inactivated ferroxidase center reduced by about ∼50% the number of apoptotic cells after TNFα-treatment, while overexpression of L-ferritin was ineffective. The data indicate that H-ferritin has an anti-apoptotic activity unrelated to its ferroxidase activity and to its capacity to modify cellular iron metabolism

Human Mitochondrial Ferritin Expressed in HeLa Cells Incorporates Iron and Affects Cellular Iron Metabolism

Mitochondrial ferritin (MtF) is a newly identified ferritin encoded by an intronless gene on chromosome 5q23.1. The mature recombinant MtF has a ferroxidase center and binds iron in vitro similarly to H-ferritin. To explore the structural and functional aspects of MtF, we expressed the following forms in HeLa cells: the MtF precursor (approximately 28 kDa), a mutant MtF precursor with a mutated ferroxidase center, a truncated MtF lacking the approximately 6-kDa mitochondrial leader sequence, and a chimeric H-ferritin with this leader sequence. The experiments show that all constructs with the leader sequence were processed into approximately 22-kDa subunits that assembled into multimeric shells electrophoretically distinct from the cytosolic ferritins. Mature MtF was found in the matrix of mitochondria, where it is a homopolymer. The wild type MtF and the mitochondrially targeted H-ferritin both incorporated the (55)Fe label in vivo. The mutant MtF with an inactivated ferroxidase center did not take up iron, nor did the truncated MtF expressed transiently in cytoplasm. Increased levels of MtF both in transient and in stable transfectants resulted in a greater retention of iron as MtF in mitochondria, a decrease in the levels of cytosolic ferritins, and up-regulation of transferrin receptor. Neither effect occurred with the mutant MtF with the inactivated ferroxidase center. Our results indicate that exogenous iron is as available to mitochondrial ferritin as it is to cytosolic ferritins and that the level of MtF expression may have profound consequences for cellular iron homeostasis

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from 0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize.This work was partially supported by MIUR grant PRIN10-11 to PA, and by Telethon grant GGP15064 to PA. FC was recipient of a Post-Doc Fellowship from University of Brescia, and was partially supported by CIB (Consorzio Italiano di Biotecnologie)

N=2 Super Yang-Mills and the XXZ spin chain

We analyse the renormalisation properties of composite operators of scalar fields in the N=2 Super Yang-Mills theory. We compute the matrix of anomalous dimensions in the planar limit at one-loop order in the 't Hooft coupling, and show that it corresponds to the Hamiltonian of an integrable XXZ spin chain with an anisotropy parameter Delta>1. We suggest that this parameter could be related to the presence of non-trivial two-form fluxes in the dual supergravity background. We find that the running of the gauge coupling does not affect the renormalization group equations for these composite operators at one-loop order, and argue that this is a general property of gauge theories which is not related to supersymmetry.Comment: 16 pages, 2 figures; v2: references added, misprints correcte

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains

This article has been accepted for publication in Protein Engineering design and Selection Published by Oxford University Press.Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5’end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli. The recombinant fusion hepcidin–ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin–ferroportin interaction in cells and also as drug-delivery agent.This work is partially financed by the Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB) and the Doctoral School of the National Institute of Applied Sciences and Technology (INSAT-Tunis) – University of Carthage