This thesis focuses on iron sulfur (Fe-S) cluster biogenesis by the ISC machinery in the mitochondrion of Trypanosoma brucei. Most of proteins in the pathway show conserved functions, while some features are distinct from their counterparts in other organisms. We also show here the essentiality of the ISC machinery in bloodstream stage despite the fact that the parasites contain the rudimentary mitochondrion in this stage. The key player for the ISC export machinery, which is indispensable in the maturation of extra-mitochondrial Fe-S proteins, shows some extraordinary phenomena which may imply the moonlighting function of the protein. I also show preliminary data of an ongoing project concerning a putative heme transporter. The results indicate role in heme uptake of the protein, but further study is required to confirm the function of the protein.

Les centres fer-soufre (Fe-S) sont des cofacteurs protéiques essentiels qui participent à un nombre important de fonctions cellulaires allant du métabolisme de l’ADN à la respiration mitochondriale. L’assemblage des centres Fe-S et leur insertion dans des protéines acceptrices requièrent l’activité d’une machinerie protéique dédiée. Bien que les protéines de la biogenèse des centres Fe-S soient conservées, plusieurs aspects fonctionnels et mécanistiques restent inconnus. Notre travail de thèse a consisté à caractériser les protéines mammifères de type A, ISCA1 et ISCA2, qui sont impliquées dans la biogenèse mitochondriales des centres Fe-S. En utilisant une approche couplant l’immunoprécipitation avec une analyse protéomique par spectrométrie de masse, plusieurs interactions protéiques d’ISCA1 et ISCA2 ont pu être identifiées. En plus d’une interaction entre ISCA1 et ISCA2, nous avons ainsi montré l’existence d’interactions spécifiques à chacune de ces protéines. Une approche de knockdown dans la souris via l’injection de virus adéno-associés, a permis de montrer l’absence de redondance fonctionnelle entre ISCA1 et ISCA2 puisque seul ISCA1 se trouve être nécessaire dans la maturation d’une catégorie de protéines à centre Fe-S. / Iron-sulfur clusters (Fe-S) are essential cofactors involved in different cellular processes ranging from DNA metabolism to respiration. Assembly of Fe-S clusters and their insertion into acceptor proteins is performed by dedicated protein machineries. Despite the high conservation from bacteria to man, different functional and mechanistic aspects of the Fe-S biogenesis remain elusive. In the present work, the function of the two mammalian A-type proteins ISCA1 and ISCA2 that are implicated in Fe-S biogenesis was investigated in vivo. First, an extensive analysis coupling immunoprecipitations and mass spectrometry led to the identification of a direct binding between ISCA1 and ISCA2 as well as specific protein partners of each protein. Furthermore, knockdown experiments in the mouse using adeno-associated virus provided clear evidence of the non-redundant function of ISCA1 and ISCA2, since only ISCA1 was shown to be required for a specific subset of mitochondrial Fe-S proteins.

L’ataxie de Friedreich est une maladie neurodégénérative sévère causée par un défaut d’expression de la frataxine (FXN), une petite protéine mitochondriale impliquée dans la biogenèse des centres fer-soufre (Fe/S), des groupement prosthétiques aux fonctions cellulaires essentielles. Chez les mammifères, il a été montré que la frataxine stimule la synthèse in vitro de centres Fe/S sur la protéine d’échaffaudage ISCU, grâce à l’augmentation de la production d’ions sulfures par le complexe NFS1-ISD11-ISCU. Cependant, le mécanisme par lequel la frataxine active la biogenèse des centres Fe/S n’a pas encore été défini. Nous avons étudié les effets de FXN sur les cinétiques de formation et de réduction des persulfures, des intermédiaires clés de la production d’ions sulfures, générés par la cystéiene désulfurase NFS1, à l’aide d’un test de détection des persulfures basé sur l’utilisation de composés synthétiques peptide-maléimide et de la spectrométrie de masse. Nous avons montré que FXN active deux réactions très similaires : la réduction du persulfure de NFS1 par des réducteurs à thiols comme le DTT, la L-cystéine et le glutathion et le transfert de soufre de NFS1 vers ISCU, conduisant à l’accumulation de persulfure sur la cystéine C104 d’ISCU. Nous avons constaté que la vitesse de réduction du persulfure d’ISCU par les thiols n’est pas affectée en présence de FXN et que ce persulfure est réduit plus lentement que celui de NFS1. Nous avons corrélé l’activation par FXN de la réduction du persulfure de NFS1 par les thiols à une stimulation de l’assemblage d’un centre Fe/S sur ISCU. Dans nos conditions expérimentales, l’atome de soufre du persulfure d’ISCU n’est pas incorporé dans le centre Fe/S synthétisé, mais nos résultats ne permettent pas d’exclure que ce persulfure puisse être réduit par une réductase dédiée, encore non identifiée. L’ensemble de nos données indiquent que le rôle de la frataxine est de contrôler la réduction du persulfure de NFS1, en augmentant les vitesses de transfert de soufre vers ISCU et de réduction du persulfure de NFS1 par les thiols. / Friedreich ataxia is a severe neurodegenerative disease caused by reduced expression of frataxin (FXN), a small mitochondrial protein involved in iron-sulfur (Fe/S) cluster biogenesis which are prostetic groups with essential cellular functions. It has been shown in vitro that mammalian FXN activates Fe/S cluster synthesis on the scaffold protein ISCU, by rising up suflide ion production by NFS1-ISD11-ISCU complex. However, the mechanism by which frataxin stimulates Fe/S cluster biogenesis has not been yet defined. We have studied the effect of FXN on the kinetics of formation and reduction of persulfides that are key intermediates of sulfide ion production generated by NFS1, using mass spectrometry and a new detection assay for persulfide based on gel-mobility shift following alkylation by maleimide-peptide compounds. We demonstrate that frataxin activates two similar reactions : sulfur transfer from cysteine desulfurase NFS1 to ISCU leading to accumulation of a persulfide on ISCUcysteine C104 and reduction of NFS1 persulfide by thiol reducers such as DTT, L-cysteine and glutathion. We have observed that FXN does not stimulate the rate of ISCU persulfide reduction by thiols and that this persulfide is reduced much more slowly than NFS1 persulfide. We have then correlated the reduction of NFS1 persulfide with Fe/S cluster assembly. Under our experimental conditions, the sulfur from ISCU persulfide is not incorporated into the Fe/S cluster. However, we cannot exclude that an as yet not identfiied reductase could reduces ISCU persulfide and trigger Fe/S cluster assembly. Overall, our data point to a regulatory function of FXN as an enhancer of persulfide reduction, stimulating the rates of sulfur transfer to ISCU and NFS1 persulfide.

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.