Action of chelators in iron-loaded cardiac cells: accessibility to intracellular labile iron and functional consequences

Glickstein, Hava; El, Rinat Ben; Link, G; Breuer, William; Konijn, Abraham M.; Hershko, C; Nick, Hanspeter; Cabantchik, Z. Ioav

doi:10.1182/blood-2006-05-020867

Cited by 178 publications

(183 citation statements)

References 43 publications

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“…The permeabilized cells were washed with permeabilization buffer (100 mM KCl, 5 mM phosphate buffer, Eagle's MEM-amino acids mix diluted 1∶500, 10 mM HEPES, 1 μM CaCl 2 , 1 mM MgSO 4 , pH 7.2) and taken to fluorescence microscopy measurements in permeabilization buffer containing 1 mM succinate. For CALG (λexc 480 nm; λ em 520 nm) (50,51) and RPA (560 nm excitation-610 nm emission) (43,44), as described elsewhere (48,50,51). DFO (1 μM) was present in all solutions during permeabilization and fluorescence measurements, to prevent RPA quenching by contaminant iron.…”

Section: Methodsmentioning

confidence: 99%

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

Zuris

Harir

Conlan

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

122

176

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MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's antidiabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster.CISD1 | diabetes | FeS cluster | oxidative stress M itoNEET (mNT) is a newly discovered target (1) of the insulin-sensitizing thiazolidinedione (TZD) class of type II diabetes drugs (2, 3), which interact with the canonical target PPARγ (4, 5). The interaction of TZD drugs with mNT has been proposed to be of therapeutic importance (1,6). This is the first iron-sulfur protein to be directly targeted by drug binding (1, 6). The protein contains two [2Fe-2S] clusters, which have been shown to be chemically labile (7). Initial characterization of mNT's redox active and pH labile [2Fe-2S] clusters show that both properties are modulated by binding of TZDs (6,8), indicating a direct interaction of the protein with the TZD drugs. A second related member of the human NEET protein family, Miner1, is structurally homologous to mNT (9). Miner1 has recently been linked to autophagy, apoptosis, and aging (10, 11), and mis-splicing is associated with a rare disease, known as Wolfram Syndrome 2 (12). Together, mNT and Miner1 represent a new class of NEET iron-sulfur (FeS) proteins characterized structurally by their unique homodimeric fold and rare 3-Cys-1-His [2Fe-2S] cluster ligand environment (6,9,13,14).Iron-sulfur (FeS) cluster-containing proteins are key players in many essential processes, such as photosynthesis, respiration, and nitrogen fixation (15, 16). They appear in various compositions and ...

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Section: Methodsmentioning

confidence: 99%

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

Zuris

Harir

Conlan

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

122

176

View full text Add to dashboard Cite

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“…For CALG-Fe(III) (1:1) complexes and for sulforhodamine loading into endosomes, cells were exposed to 50 M to 100 M complex in DMEM-HEPES for 30 minutes at 37°C and washed extensively with the same probe-free medium and subsequently with HEPES-buffered saline (HBS; 20 mM HEPES, 135 mM NaCl; pH 7.4). 20,21 RPA was loaded into mitochondria as described in earlier studies. [21][22][23] Histone-CALG.…”

Section: Methodsmentioning

confidence: 99%

“…20,21 RPA was loaded into mitochondria as described in earlier studies. [21][22][23] Histone-CALG. For loading into the nucleus, CALG was coupled to core histones from calf thymus (H) 7.2 mg/mL, in 50 mM Na-MES, 100 mM NaCl, pH 5.5, containing 1.25 mM CALG:Cobalt (Co protects CALG metal-binding groups).…”

Section: Methodsmentioning

confidence: 99%

“…H-CALG-Fe penetrates the plasma membrane and accumulates initially in the cytosol and subsequently in the nucleus, particularly in nucleolus-like sites. 21 H-CALG-Fe was used subsaturated with iron so that its accumulation in the nuclei could be followed via changes in the residual fluorescence of the unquenched fraction of the probe (revealed by chelation; Figure 3A). Loading of RPA into mitochondria was apparent from the characteristic perinuclear staining pattern ( Figure 3C), from colocalization with other potentiometric probes and sensitivity to protonophores.…”

Section: Transfer Of Iron From Extracellular Dfp-iron Complexes To MImentioning

confidence: 99%

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Redistribution of accumulated cell iron: a modality of chelation with therapeutic implications

Sohn

Breuer

Münnich

et al. 2008

Blood

Self Cite

161

149

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Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. Deferiprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissuechelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation. IntroductionThe pathologic effects of iron accumulation in tissue are recognized in diseases of systemic iron overload, in which the liver, heart, and endocrine glands are the principal affected organs. 1 At the cellular level, labile iron begins to rise once the intracellular capacity for iron storage is surpassed, leading to catalytic formation of reactive oxygen species (ROS) that ultimately overwhelm the cellular antioxidant defense mechanisms and lead to cell damage. 2 In recent years, several pathologies have been shown to be associated with specific defects in cellular iron metabolism that do not give rise to conspicuous systemic iron overload. 3 In the neuromuscular disorder Friedreich ataxia, the deficiency of the iron-chaperone protein frataxin is thought to lead to mitochondrial iron accumulation due to improper processing of iron for heme and iron-sulfur-cluster formation. 4 In the group of neuromuscular disorders termed NBIA (neurodegeneration with brain iron accumulation), a deficiency in pantothenate kinase (PKAN-2), a key enzyme in coenzyme A synthesis, 5 leads to iron deposition and ensuing brain damage by still-unresolved mechanisms. 6 In hereditary X-linked sideroblastic anemia, impaired heme synthesis causes mitochondrial iron accumulation and siderosis in erythroid cells. 3 However, an extreme example of systemic misdistribution of iron is encountered in anemia of chronic disease (ACD), where iron accumulates in reticuloendothelial cells responsible for recycling aged erythrocytes, presumably due to d...

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“…The permeabilized cells were washed with permeabilization buffer (100 mM KCl, 5 mM Na 2 HPO 4 , Eagle's minimal essential medium-amino acids mix diluted 1:500, 10 mM HEPES, 1 µM CaCl 2 , and 1 mM MgSO 4 , pH 7.2) and used for fluorescence microscopy measurements in permeabilization buffer containing 1 mM succinate. For CALG (480-nm excitation; 520-nm emission) (Espósito et al, 2002;Glickstein et al, 2006) and RPA (560-nm excitation; 610-nm emission) (Petrat et al, 2002;Rauen et al, 2003), as described elsewhere (Espósito et al, 2002;Glickstein et al, 2005Glickstein et al, , 2006. Desferrioxamine (1 µM) was present in all solutions during permeabilization and fluorescence measurements to prevent RPA quenching by contaminant Fe.…”

Section: Fluorescence Measurementmentioning

confidence: 99%

Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism

et al. 2012

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The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

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Action of chelators in iron-loaded cardiac cells: accessibility to intracellular labile iron and functional consequences

Cited by 178 publications

References 43 publications

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

Redistribution of accumulated cell iron: a modality of chelation with therapeutic implications

Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism

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