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At least 19 recordsLinked to original sources
PubMed2002

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization.

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis.

Animals↗
PubMed2004

Candida albicans lacking the frataxin homologue: a relevant yeast model for studying the role of frataxin.

We cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans. CaYFH1 was expressed in Deltayfh1 Saccharomyces cerevisiae cells, where it compensated for all the phenotypes tested except for the lack of cytochromes. Double DeltaCayfh1/DeltaCayfh1 mutant had severe defective growth, accumulated iron in their mitochondria, lacked aconitase and succinate dehydrogenase activity and had defective respiration. The reductive, siderophore and haem uptake systems were constitutively induced and the cells excreted flavins, thus behaving like iron-deprived wild-type cells. Mutant cells accumulated reactive oxygen species and were hypersensitive to oxidative stress, but not to iron. Cytochromes were less abundant in mutants than in wild-type cells, but this did not result from defective haem synthesis. The low cytochrome concentration in mutant cells was comparable to that of iron-deprived wild-type cells. Mitochondrial iron was still available for haem synthesis in DeltaCayfh1/DeltaCayfh1 cells, in contrast to S. cerevisaeDeltayfh1 cells. CaYFH1 transcription was strongly induced by iron, which is consistent with a role of CaYfh1 in iron storage. Iron also regulated transcription of CaHEM14 (encoding protoporphyrinogen oxidase) but not that of CaHEM15 (encoding ferrochelatase). There are thus profound differences between S. cerevisiae and C. albicans in terms of haem synthesis, cytochrome turnover and the role of frataxin in these processes.

Candida albicans↗
PubMed2002

Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of frataxin, a conserved mitochondrial protein of unknown function. Mitochondrial iron accumulation, loss of iron-sulfur cluster-containing enzymes and increased oxidative damage occur in yeast and mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA patients, suggesting that frataxin may be involved in export of iron from the mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative damage. We have previously shown that yeast frataxin has structural and functional features of an iron storage protein. In this study we have investigated the function of human frataxin in Escherichia coli and Saccharomyces cerevisiae. When expressed in E.coli, the mature form of human frataxin assembles into a stable homopolymer that can bind approximately 10 atoms of iron per molecule of frataxin. The iron-loaded homopolymer can be detected on non-denaturing gels by either protein or iron staining demonstrating a stable association between frataxin and iron. As analyzed by gel filtration and electron microscopy, the homopolymer consists of globular particles of approximately 1 MDa and ordered rod-shaped polymers of these particles that accumulate small electron-dense cores. When the human frataxin precursor is expressed in S.cerevisiae, the mitochondrially generated mature form is separated by gel filtration into monomer and a high molecular weight pool of >600 kDa. A high molecular weight pool of frataxin is also present in mouse heart indicating that frataxin can assemble under native conditions. In radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation with approximately five atoms of (55)Fe bound per molecule. These findings suggest that FRDA results from decreased mitochondrial iron storage due to frataxin deficiency which may impair iron metabolism, promote oxidative damage and lead to progressive iron accumulation.

Animals↗
PubMed2005

Supramolecular assemblies of human frataxin are formed via subunit-subunit interactions mediated by a non-conserved amino-terminal region.

The mitochondrial protein frataxin is emerging as a novel mechanism to promote iron metabolism while also providing anti-oxidant protection. Recombinant frataxin proteins from different species are able to form large molecular assemblies that store Fe(III) as a stable mineral in vitro. Furthermore, monomeric and assembled forms of frataxin donate Fe(II) to the Fe-S cluster scaffold protein IscU, [3Fe-4S]1+ aconitase, and ferrochelatase in vitro. However, little is known about the speciation of frataxin in vivo, and the physiologically relevant form(s) of the protein remains undefined. Here, we report that human heart mitochondria contain frataxin species of increasing negative surface charge and molecular mass, ranging from monomer to polymers of >1 MDa. Moreover, we show that the main partner protein of frataxin, IscU, binds in a stable manner to frataxin oligomers. These results suggest that assembly is a physiologic property of frataxin. Biochemical analyses further reveal that, unlike the prokaryotic and yeast frataxin homologues, which require iron-protein interactions for assembly, human frataxin uses stable subunit-subunit interactions involving a non-conserved amino-terminal region. We propose that human frataxin is a modular protein that depends on self-assembly to accomplish its diverse functions.

Biopolymers↗
PubMed2006

Monomeric yeast frataxin is an iron-binding protein.

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

Chromatography, Gel↗
PubMed2001

Frataxin deficiency enhances apoptosis in cells differentiating into neuroectoderm.

Deficiency of the mitochondrial matrix protein frataxin causes Friedreich ataxia. Frataxin function is believed to be related to mitochondrial iron metabolism and free radical production. In Friedreich ataxia, loss of dorsal root ganglia neurons occurs early in life, suggesting a developmental process. In addition, frataxin knockout mice die during embryonic life, further suggesting that frataxin is necessary for normal development. In this study we examine the role of frataxin in neuronal differentiation by using the P19 embryonic carcinoma cell line as a model system. We produced stably transfected clones with antisense or sense frataxin constructs. During retinoic acid-induced neurogenesis of frataxin-deficient cells there was a striking rise in cell death, while cell division remained unaffected. However, frataxin deficiency does not affect cell survival in cells induced to differentiate into cardiomyocytes. Frataxin deficiency enhances apoptosis of retinoic acid-stimulated cells, and the number of neuronal-like cells expressing MAP2 was dramatically reduced in these clones. In addition, we found that antisense clones induced to differentiate into neuroectoderm with retinoic acid have increased production of reactive oxygen species, and that only cells non-committed to the neuronal lineages could be rescued by the addition of the antioxidant N-acetyl-cysteine (NAC). However, NAC treatment had no effect in increasing the number of terminally differentiated neuronal-like cells in frataxin-deficient clones. Our results suggest that frataxin deficiency may render cells susceptible to apoptosis after exposure to appropriate stimuli.

Acetylcysteine↗
PubMed2003

Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins.

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM] and isothermal titration calorimetry (ITC) [K(D)(Fe(3+)) approximately 10.2 microM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774-8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.

Escherichia coli↗
PubMed2003

Iron metabolism in mice with partial frataxin deficiency.

Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene. The yeast frataxin homologue (yfh1p) is required for cellular respiration. Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity. Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development. Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype. Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx(+/-)). Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx(+/-) mice. Response to parenteral iron challenge was not different between Fx(+/-) mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx(+/-) mice. Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice. HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin. By breeding Hfe(-/-) with Fx(+/-) mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele. Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice. However, they did not develop any neurological symptoms. Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model. In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.

Animals↗
PubMed2005

Mitochondrial iron detoxification is a primary function of frataxin that limits oxidative damage and preserves cell longevity.

Friedreich ataxia is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin. Although frataxin is ubiquitously expressed, frataxin deficiency leads to a selective loss of dorsal root ganglia neurons, cardiomyocytes and pancreatic beta cells. How frataxin normally promotes survival of these particular cells is the subject of intense debate. The predominant view is that frataxin sustains mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron-sulfur cluster (ISC) assembly and repair. We have proposed that frataxin not only promotes the biogenesis of iron-containing enzymes, but also detoxifies surplus iron thereby affording a critical anti-oxidant mechanism. These two functions have been difficult to tease apart, however, and the physiologic role of iron detoxification by frataxin has not yet been demonstrated in vivo. Here, we describe mutations that specifically impair the ferroxidation or mineralization activity of yeast frataxin, which are necessary for iron detoxification but do not affect the iron chaperone function of the protein. These mutations increase the sensitivity of yeast cells to oxidative stress, shortening chronological life span and precluding survival in the absence of the anti-oxidant enzyme superoxide dismutase. Thus, the role of frataxin is not limited to promoting ISC assembly or heme synthesis. Iron detoxification is another function of frataxin relevant to anti-oxidant defense and cell longevity that could play a critical role in the metabolically demanding environment of non-dividing neuronal, cardiac and pancreatic beta cells.

Amino Acids↗
PubMed2004

Yeast frataxin solution structure, iron binding, and ferrochelatase interaction.

The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis. Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively. We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase. While the protein's alpha-beta-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion. In addition, NMR spectroscopy was used to identify frataxin amino acids that are perturbed by the presence of iron. Conserved acidic residues in the helix 1-strand 1 protein region undergo amide chemical shift changes in the presence of Fe(II), indicating a possible iron-binding site on frataxin. NMR spectroscopy was further used to identify the intermolecular binding interface between ferrochelatase and frataxin. Ferrochelatase appears to bind to frataxin's helical plane in a manner that includes its iron-binding interface.

Binding Sites↗
PubMed2002

A structural approach to understanding the iron-binding properties of phylogenetically different frataxins.

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin--as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms iron-promoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the beta sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.

Amino Acid Sequence↗
PubMed2002

Frataxin deficiency and mitochondrial dysfunction.

Friedreich ataxia (FA) is an inherited recessive disorder characterized by progressive neurological disability and heart abnormalities. The Friedreich ataxia gene (FRDA) encodes a small mitochondrial protein, frataxin, which is produced in insufficient amounts in the disease as a consequence of a GAA triplet repeat expansion in the first intron of the gene. Frataxin deficiency leads to excessive free radical production, dysfunction of Fe-S center containing enzymes (in particular respiratory complexes I, II and III, and aconitase), and progressive iron accumulation in mitochondria. Frataxin may be a mitochondrial iron-binding protein that prevents this metal from participating in Fenton chemistry to generate toxic hydroxyl radicals. We investigated whether frataxin deficiency may in addition interfere with signaling pathways. First, we showed that exposure of FA fibroblasts to iron fails to produce the normally observed increase in expression of the stress defense protein manganese superoxide dismutase. This impaired induction involves a nuclear factor-kappaB-independent pathway that does not require free radical signaling intermediates. We also examined the role of frataxin in neuronal differentiation by using stably transfected clones of P19 embryonic carcinoma cells with antisense or sense frataxin constructs. We found that during retinoic acid-induced neurogenesis frataxin deficiency enhances apoptosis and reduces the number of terminally differentiated neuronal-like cells. The addition of the antioxidant N-acetyl-cysteine only rescues cells non-committed to the neuronal lineage, indicating that frataxin deficiency impairs differentiation mechanisms and survival responses through different mechanisms. Both studies suggest that some abnormalities in frataxin-deficient cells are related to free radical independent signaling pathways.

Journal Article↗
PubMed2005

Molecular and functional characterization of a human frataxin mutation found in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is associated with marked genetic and phenotypic heterogeneity. Pathogenic mutations in the 10 hypertrophic cardiomyopathy-associated sarcomeric genes cause autosomal dominant disease as a rule, although recessive disease has been reported. Cardiac hypertrophy is also a hallmark of Friedreich ataxia, an autosomal recessive disease caused by deficiency of the mitochondrial protein frataxin. We hypothesized that heterozygous mutations in frataxin may mimic or modify hypertrophic cardiomyopathy. Using DHPLC and DNA sequencing, we identified the novel R40C-frataxin mutation in a patient who also harbored a previously reported R810H-myosin binding protein C mutation. The R810H mutation is reported to cause hypertrophic cardiomyopathy only in the setting of homozygosity or compound heterozygosity with another sarcomeric mutation. Site-directed mutagenesis and in vitro and in vivo analysis enabled functional characterization of the mutant frataxin protein. R40C-frataxin protein is not cleaved to the mature form in vitro and shows delayed kinetics of cleavage by isolated mouse mitochondria. Yeast cells expressing R40C-frataxin demonstrated increased sensitivity to oxidative stress and abnormal accumulation of precursor frataxin protein. These data indicate that frataxin deficiency may have contributed to this patient's particular phenotype. Furthermore, these findings suggest that mutations altering myocyte energetics may act in synergy with sarcomeric mutations to cause hypertrophic cardiomyopathy.

Cardiomyopathy, Hypertrophic↗
PubMed1999

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.

Amino Acid Sequence↗
PubMed2003

Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells.

Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. We analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, we did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.

Amino Acids, Sulfur↗
PubMed2005

Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2.

Friedreich's ataxia is a recessive neurodegenerative disease due to insufficient expression of the mitochondrial protein frataxin. Although it has been shown that frataxin is involved in the control of intracellular iron metabolism, by interfering with the mitochondrial biosynthesis of proteins with iron/sulphur (Fe/S) clusters its role has not been well established. We studied frataxin protein and mRNA expression and localisation during cellular differentiation. We used the human colon adenocarcinoma cell line Caco-2, as it is considered a good model for intestinal epithelial differentiation and the study of intestinal iron metabolism. Here we report that the protein, but not the mRNA frataxin levels, increase during the enterocyte-like differentiation of Caco-2 cells, as well as in in-vivo-differentiated enterocytes at the upper half of the crypt-villus axis. Furthermore, subcellular fractionation and double immunostaining, followed by confocal analysis, reveal that frataxin localisation changes during Caco-2 cell differentiation. In particular, we found an extramitochondrial localisation of frataxin in differentiated cells. Finally, we demonstrate a physical interaction between extramitochondrial frataxin and IscU1, a cytoplasmic isoform of the human Fe/S cluster assembly machinery. Based on our data, we postulate that frataxin could be involved in the biosynthesis of iron-sulphur proteins not only within the mitochondria, but also in the extramitochondrial compartment. These findings might be of relevance for the understanding of both the pathogenesis of Friedreich's ataxia and the basic mechanism of Fe/S cluster biosynthesis.

Caco-2 Cells↗
PubMed2003

Cisplatin may induce frataxin expression.

Cisplatin is a widely used drug in cancer chemotherapy and resistance to cisplatin is a major limitation for its successful application. Intracellular inactivation of cisplatin and detoxification of reactive oxygen species (ROS) by glutathione (a crucial cellular antioxidant) is a mechanism for cisplatin resistance. During cDNA microarray analyses of differential gene expression between a cisplatin-resistant A2780CP70 human ovarian carcinoma cell line and its parental A2780 cell line, we discovered that frataxin gene expression was frequently overexpressed in the cisplatin-resistant variant. Decreased expression of frataxin protein is associated with Friedreich's ataxia (FRDA) which is a neurodegenerative disease involving ROS-mediated cellular damage. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols. To exploit potential involvement of frataxin gene in the development of resistance to cisplatin, we compared the levels of frataxin gene and protein in the cisplatin-resistant A2780CP70 ovarian carcinoma cell line and its parental A2780 cell line. We found that frataxin mRNA and protein expressions were elevated in the cisplatin-resistant cells. Our results suggest a potential role for cisplatin as an inducer of frataxin expression and implies that this gene may be a potential target for modulating the response to cisplatin. This is the first report showing an association between frataxin expression and cisplatin resistance.

Cisplatin↗