Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/NFS1/ISD11 Iron-Sulfur Assembly Complex

Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis.. Fe-S cluster biosynthesis

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

BACKGROUND:Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation. METHODOLOGY:We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients. CONCLUSIONS:These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency

Measurement of the B0^0 and B+^+ meson lifetimes with fully reconstructed hadronic final states

The B0 and B+ meson lifetimes have been measured in e+e- annihilation data collected in 1999 and 2000 with the BABAR detector at center-of-mass energies near the Upsilon(4S) resonance. Events are selected in which one B meson is fully reconstructed in a hadronic final state while the second B meson is reconstructed inclusively. A combined fit to the B0 and the B+ decay time difference distributions yields tau_{B0} = 1.546 +/- 0.032 (stat) +/- 0.022(syst) ps, tau_{B+} = 1.673 +/- 0.032 (stat) +/- 0.023 (syst) ps and tau_{B+} / tau_{B0} = 1.082 +/- 0.026 (stat) +/- 0.012 (syst

La frataxine, protéine déficiente dans l’ataxie de Friedreich : comprendre sa maturation, sa fonction et les conséquences physiopathologiques de son déficit

L’ataxie de Friedreich (AF) est une maladie génétique récessive neurodégénérative due à la perte de fonction d’une protéine mitochondriale, la frataxine (FXN). La mutation majoritaire est une expansion trinucléotidique GAA dans le premier intron du gène de la frataxine (FXN), à l’origine d’une diminution sévère de la transcription. La fonction exacte de la FXN n’a pas été élucidée, mais son absence cause un déficit des enzymes à noyau Fe-S, une accumulation mitochondriale de fer et une sensibilité au stress oxydant. (1) La FXN est synthétisée sous forme d’un précurseur qui sera importé dans la mitochondrie. Sa maturation implique deux clivages. J’ai pu démontrer par spectrométrie de masse (SM) que le site final de maturation se situe entre les acides aminés 80 et 81. (2) Nous avons identifié chez un patient, une mutation R26G dans l’adressage mitochondrial de la FXN perturbant l’import dans la mitochondrie. (3) J’ai participé au développement de modèles cellulaires reproduisant les caractéristiques biochimiques de l’AF. Ces modèles sont basés sur l’expression de formes mutées de FXN humaine (G130V et I154F) dans des fibroblastes murins où le gène FXN a été inactivé. (4) Par des études d’interactions combinées à la SM, nous avons déterminé que les interacteurs de la FXN sont ISCU, NFS1 et ISD11, impliqués dans la biosynthèse des centres Fe-S. Par co-purification des quatre protéines, nous avons isolé un complexe FXN/ISCU/NFS1/ISD11. Par une approche in vivo avec des cellules en culture, nous avons montré que la déstabilisation spécifique du complexe induisait un phénotype très sévère. Ces données démontrent le rôle crucial de la FXN dans l’assemblage des centres Fe-S.Friedreich ataxia (FRDA) is a recessive neurodegenerative disease due to a loss of function of a mitochondrial protein called frataxin (FXN). The most common mutation is a GAA triplet expansion in the first intron of FXN gene causing a severe transcription alteration. The exact function of FXN is unclear but its deficiency is associated to iron-sulfur enzymes deficit, mitochondrial iron accumulation and susceptibility to oxidative stress. (1) FXN is synthesized as a precursor targeted to mitochondria. Its maturation involves two cleavages. I demonstrate by mass spectrometry (MS) that the final maturation site is localized between amino acids 80 and 81. (2) We identified in a patient, a R26G mutation in the mitochondrial targeting sequence of FXN impairing the mitochondrial import. (3) I participate to the development of cellular models that reproduce the FRDA biochemical features. These models are based on expression of mutated human FXN forms (G130V and I154F) in murine fibroblasts where the endogenous FXN gene was inactivated. (4) By interaction studies coupled to MS, we determined that FXN interacts with ISCU, NFS1, ISD11, three components of the iron-sulfur cluster biosynthesis. By co-purification tests, we isolate a FXN/ISCU/NFS1/ISD11 quaternary complex. By an in vivo approach on cell cultures, we show that destabilizing the formation of the quaternary complex causes a severe phenotype. These results demonstrate a crucial role of FXN in the iron-sulfur cluster biosynthesis

Frataxin, the protein deficient in Friedreich ataxia,towards a better understanding of its maturation, its function and the pathophysiological consequences of its deficit

L'ataxie de Friedreich (AF) est une maladie génétique récessive neurodégénérative due à la perte de fonction d'une protéine mitochondriale, la frataxine (FXN). La mutation majoritaire est une expansion trinucléotidique GAA dans le premier intron du gène de la frataxine (FXN), à l'origine d'une diminution sévère de la transcription. La fonction exacte de la FXN n a pas été élucidée, mais son absence cause un déficit des enzymes à noyau Fe-S, une accumulation mitochondriale de fer et une sensibilité au stress oxydant. (1) La FXN est synthétisée sous forme d'un précurseur qui sera importé dans la mitochondrie. Sa maturation implique deux clivages. J ai pu démontrer par spectrométrie de masse (SM) que le site final de maturation se situe entre les acides aminés 80 et 81. (2) Nous avons identifié chez un patient, une mutation R26G dans l'adressage mitochondrial de la FXN perturbant l'import dans la mitochondrie. (3) J ai participé au développement de modèles cellulaires reproduisant les caractéristiques biochimiques de l'AF. Ces modèles sont basés sur l'expression de formes mutées de FXN humaine (G130V et I154F) dans des fibroblastes murins où le gène FXN a été inactivé. (4) Par des études d'interactions combinées à la SM, nous avons déterminé que les interacteurs de la FXN sont ISCU, NFS1 et ISD11, impliqués dans la biosynthèse des centres Fe-S. Par co-purification des quatre protéines, nous avons isolé un complexe FXN/ISCU/NFS1/ISD11. Par une approche in vivo avec des cellules en culture, nous avons montré que la déstabilisation spécifique du complexe induisait un phénotype très sévère. Ces données démontrent le rôle crucial de la FXN dans l'assemblage des centres Fe-S.Friedreich ataxia (FRDA) is a recessive neurodegenerative disease due to a loss of function of a mitochondrial protein called frataxin (FXN). The most common mutation is a GAA triplet expansion in the first intron of FXN gene causing a severe transcription alteration. The exact function of FXN is unclear but its deficiency is associated to iron-sulfur enzymes deficit, mitochondrial iron accumulation and susceptibility to oxidative stress. (1) FXN is synthesized as a precursor targeted to mitochondria. Its maturation involves two cleavages. I demonstrate by mass spectrometry (MS) that the final maturation site is localized between amino acids 80 and 81. (2) We identified in a patient, a R26G mutation in the mitochondrial targeting sequence of FXN impairing the mitochondrial import. (3) I participate to the development of cellular models that reproduce the FRDA biochemical features. These models are based on expression of mutated human FXN forms (G130V and I154F) in murine fibroblasts where the endogenous FXN gene was inactivated. (4) By interaction studies coupled to MS, we determined that FXN interacts with ISCU, NFS1, ISD11, three components of the iron-sulfur cluster biosynthesis. By co-purification tests, we isolate a FXN/ISCU/NFS1/ISD11 quaternary complex. By an in vivo approach on cell cultures, we show that destabilizing the formation of the quaternary complex causes a severe phenotype. These results demonstrate a crucial role of FXN in the iron-sulfur cluster biosynthesis

Frataxin, the protein deficient in Friedreich ataxia,towards a better understanding of its maturation, its function and the pathophysiological consequences of its deficit

L'ataxie de Friedreich (AF) est une maladie génétique récessive neurodégénérative due à la perte de fonction d'une protéine mitochondriale, la frataxine (FXN). La mutation majoritaire est une expansion trinucléotidique GAA dans le premier intron du gène dFriedreich ataxia (FRDA) is a recessive neurodegenerative disease due to a loss of function of a mitochondrial protein called frataxin (FXN). The most common mutation is a GAA triplet expansion in the first intron of FXN gene causing a severe transcripti

The in vivo mitochondrial two-step maturation of human frataxin.

International audienceDeficiency in the nuclear-encoded mitochondrial protein frataxin causes Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associating spinocerebellar ataxia and cardiomyopathy. Although the exact function of frataxin is still a matter of debate, it is widely accepted that frataxin is a mitochondrial iron chaperone involved in iron-sulfur cluster and heme biosynthesis. Frataxin is synthesized as a precursor polypeptide, directed to the mitochondrial matrix where it is proteolytically cleaved by the mitochondrial processing peptidase to the mature form via a processing intermediate. The mature form was initially reported to be encoded by amino acids 56-210 (m(56)-FXN). However, two independent reports have challenged these studies describing two different forms encoded by amino acids 78-210 (m(78)-FXN) and 81-210 (m(81)-FXN). Here, we provide evidence that mature human frataxin corresponds to m(81)-FXN, and can rescue the lethal phenotype of fibroblasts completely deleted for frataxin. Furthermore, our data demonstrate that the migration profile of frataxin depends on the experimental conditions, a behavior which most likely contributed to the confusion concerning the endogenous mature frataxin. Interestingly, we show that m(56)-FXN and m(78)-FXN can be generated when the normal maturation process of frataxin is impaired, although the physiological relevance is not clear. Furthermore, we determine that the d-FXN form, previously reported to be a degradation product, corresponds to m(78)-FXN. Finally, we demonstrate that all frataxin isoforms are generated and localized within the mitochondria. The clear identification of the N-terminus of mature FXN is an important step for designing therapeutic approaches for FRDA based on frataxin replacement

Frataxin is essential for extramitochondrial Fe S cluster proteins in mammalian tissues.

Friedreich ataxia, the most common recessive ataxia, is caused by the deficiency of the mitochondrial protein frataxin (Fxn), an iron chaperone involved in the assembly of Fe-S clusters (ISC). In yeast, mitochondria play a central role for all Fe-S proteins, independently of their subcellular localization. In mammalian cells, this central role of mitochondria remains controversial as an independent cytosolic ISC assembly machinery has been suggested. In the present work, we show that three extramitochondrial Fe-S proteins (xanthine oxido-reductase, glutamine phosphoribosylpyrophosphate amidotransferase and Nth1) are affected in Fxn-deleted mouse tissues. Furthermore, we show that Fxn is strictly localized to the mitochondria, excluding the presence of a cytosolic pool of Fxn in normal adult tissues. Together, these results demonstrate that in mammals, Fxn and mitochondria play a cardinal role in the maturation of extramitochondrial Fe-S proteins. The Fe-S scaffold protein IscU progressively decreases in Fxn-deleted tissues, further contributing to the impairment of Fe-S proteins. These results thus provide new cellular pathways that may contribute to molecular mechanisms of the disease

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.

International audienceMammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist