Reduced expression of frataxin extends the lifespan of Caenorhabditis elegans

Defects in the expression of the mitochondrial protein frataxin cause Friedreich's ataxia, an hereditary neurodegenerative syndrome characterized by progressive ataxia and associated with reduced life expectancy in humans. Homozygous inactivation of the frataxin gene results in embryonic lethality in mice, suggesting that frataxin is required for organismic survival. Intriguingly, the inactivation of many mitochondrial genes in the nematode Caenorhabditis elegans by RNAi extends lifespan. We therefore investigated whether inactivation of frataxin by RNAi-mediated suppression of the frataxin homolog gene (frh-1) would also prolong lifespan in the nematode. Frataxin-deficient animals have a small body size, reduced fertility and altered responses to oxidative stress. Importantly, frataxin suppression by RNAi significantly extends lifespan in C. elegans.     

Exploring frataxin function

Frataxin is a nuclear-encoded mitochondrial protein highly conserved in prokaryotes and eukaryotes. Its deficiency was initially described as the phenotype of Friedreich's ataxia, an autosomal recessive disease in humans. Although several functions have been described for frataxin, that is, involvement in Fe-S cluster and heme synthesis, energy conversion and oxidative phosphorylation, iron handling and response to oxidative damage, its precise function remains unclear. Although there is a general consensus on the participation of frataxin in the maintenance of cellular iron homeostasis and in iron metabolism, this protein may have other specific functions in different tissues and organisms.     

Overexpression of frataxin in the mitochondria increases resistance to oxidative stress and extends lifespan in Drosophila

In Friedreich's ataxia, reduction of the mitochondria protein frataxin results in the accumulation of iron and reactive oxygen species, which leads to oxidative damage, neurodegeneration and a diminished lifespan. Recent studies propose that frataxin might play a role in the antioxidative process. Here we show that overexpression of Drosophila frataxin in the mitochondria of female transgenic animals increases antioxidant capability, resistance to oxidative stress insults, and longevity. This suggests that Drosophila frataxin may function to protect the mitochondria from oxidative stresses and the ensuing cellular damage.     

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.     

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.     

Partial conservation of functions between eukaryotic frataxin and the Escherichia coli frataxin homolog CyaY

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.     

Assembly of human frataxin is a mechanism for detoxifying redox-active iron

Mitochondrial function depends on a continuous supply of iron to the iron-sulfur cluster (ISC) and heme biosynthetic pathways as well as on the ability to prevent iron-catalyzed oxidative damage. The mitochondrial protein frataxin plays a key role in these processes by a novel mechanism that remains to be fully elucidated. Recombinant yeast and human frataxin are able to self-associate in large molecular assemblies that bind and store iron as a ferrihydrite mineral. Moreover, either single monomers or polymers of human frataxin have been shown to serve as donors of Fe(II) to ISC scaffold proteins, oxidatively inactivated [3Fe-4S](+) aconitase, and ferrochelatase. These results suggest that frataxin can use different molecular forms to accomplish its functions. Here, stable monomeric and assembled forms of human frataxin purified from Escherichia coli have provided a tool for testing this hypothesis at the biochemical level. We show that human frataxin can enhance the availability of Fe(II) in monomeric or assembled form. However, the monomer is unable to prevent iron-catalyzed radical reactions and the formation of insoluble ferric iron oxides. In contrast, the assembled protein has ferroxidase activity and detoxifies redox-active iron by sequestering it in a protein-protected compartment.     

Evidence for a role of frataxin in pancreatic islets isolated from multi-organ donors with and without type 2 diabetes mellitus

Frataxin (FXN) is a mitochondrial protein involved in iron metabolism and in the modulation of reactive oxygen and/or nitrogen species production. No information is currently available as for the role of frataxin in isolated human pancreatic islets. We studied islets from pancreases of multi-organ donors with (T2DM) and without (Ctrl) Type 2 diabetes mellitus. In these islets, we determined FXN gene and protein expression by qualitative and quantitative Real-Time RT-PCR, nitrotyrosine concentration, and insulin release in response to glucose stimulation (SI). FXN gene and protein were expressed in human islets, though the level of expression was much lower in T2DM islets. The latter also had lower insulin release and higher concentration of nitrotyrosine. A positive correlation was apparent between SI and FXN gene expression, while a negative correlation was found between nitrotyrosine islet concentration and FXN expression. Transfection of Ctrl islets with siRNA FXN caused reduction of FXN expression, increase of nitrotyrosine concentration, and reduction of insulin release. In conclusion, in human pancreatic islets FXN contributes to regulation of oxidative stress and insulin release in response to glucose. In islets from T2DM patients FXN expression is reduced while oxidative stress is increased and insulin release in response to glucose impaired.     

Deferiprone and idebenone rescue frataxin depletion phenotypes in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA), the most common inherited ataxia, is a neurodegenerative disease caused by a reduction in the levels of the mitochondrial protein frataxin, the function of which remains a controversial matter. Several therapeutic approaches are being developed to increase frataxin expression and reduce the intramitochondrial iron aggregates and oxidative damage found in this disease. In this study, we tested separately the response of a Drosophila RNAi model of FRDA ( Llorens et al., 2007) to treatment with the iron chelator deferiprone (DFP) and the antioxidant idebenone (IDE), which are both in clinical trials. The FRDA flies have a shortened life span and impaired motor coordination, and these phenotypes are more pronounced in oxidative stress conditions. In addition, under hyperoxia, the activity of the mitochondrial enzyme aconitase is strongly reduced in the FRDA flies. This study reports that DFP and IDE improve the life span and motor ability of frataxin-depleted flies. We show that DFP eliminates the excess of labile iron in the mitochondria and thus prevents the toxicity induced by iron accumulation. IDE treatment rescues aconitase activity in hyperoxic conditions. These results validate the use of our Drosophila model of FRDA to screen for therapeutic molecules to treat this disease.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Assembly of the iron-binding protein frataxin in Saccharomyces cerevisiae responds to dynamic changes in mitochondrial iron influx and stress level

Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to > or = 24-mer, independent of interactions between Yfh1 and its major iron-binding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.     

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

The structure and function of frataxin

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich's ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin's specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein's role in different cellular pathways.     

Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites

CyaY is the bacterial ortholog of frataxin, a small mitochondrial iron binding protein thought to be involved in iron sulphur cluster formation. Loss of frataxin function leads to the neurodegenerative disorder Friedreich's ataxia. We have solved the solution structure of CyaY and used the structural information to map iron binding onto the protein surface. Comparison of the behavior of wild-type CyaY with that of a mutant indicates that specific binding with a defined stoichiometry does not require aggregation and that the main binding site, which hosts both Fe(2+) and Fe(3+), occupies a highly anionic surface of the molecule. This function is conserved across species since the corresponding region of human frataxin is also able to bind iron, albeit with weaker affinity. The presence of secondary binding sites on CyaY, but not on frataxin, hints at a possible polymerization mechanism. We suggest mutations that may provide further insights into the frataxin function.     

A pool of extramitochondrial frataxin that promotes cell survival

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.     

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.     

An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1

Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.     

Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.