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.

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Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...

Des modifications dans le gène codant pour la sous-unité II du cytochrome c oxydase du génome mâle (Mcox2) ont été recensées chez des espèces de bivalves présentant un mode unique de transmission mitochondriale nommé double transmission uniparentale (DUI). Dans la DUI, les mitochondries paternelles (et leur ADNmt mâle) ainsi que les mitochondries maternelles (et leur ADNmt femelle) sont transmises aux descendants mâles. Scrobicularia plana, une espèce de bivalves présentant ce modèle d'hérédité, possède une insertion importante d'environ 4,8 kb dans son gène Mcox2 qui ne change pas le cadre de lecture et qui est traduite en un polypeptide de 1 892 acides aminés, ce qui en fait la plus grande protéine COX2 connue à ce jour chez les métazoaires. L’objectif de cette étude était de caractériser l'évolution et la fonction potentielle de l'insertion dans Mcox2 chez S. plana par RT-PCR, tests immunologiques et analyses bio-informatiques. L'insertion est présente parmi les individus de différentes populations, contient des variations dans la longueur de sa séquence, est riche en zones de désordre intrinsèque et évolue sous sélection purificatrice. La longue insertion pourrait modifier la structure 3D du complexe IV de la chaîne de transport d'électrons (CTE), affectant sa fonction dans la phosphorylation oxydative (OXPHOS) ce qui pourrait expliquer les faibles taux d'OXPHOS observés dans les mitochondries mâles des bivalves à DUI. L'insertion pourrait également modifier le métabolisme mitochondrial mâle en interagissant avec d'autres complexes de la CTE et avec l'ATP synthase. Comme pour les autres modifications de Mcox2 chez les bivalves à DUI, un rôle potentiel dans la détermination du sexe peut être prédit pour MCOX2 chez S. plana. / Modifications in the cytochrome c oxidase subunit II gene of male-transmitted genome (Mcox2) have been found in some bivalve species that exhibit a unique mode of mitochondrial transmission named doubly uniparental inheritance (DUI). In DUI, paternal mitochondria (and their male mtDNA) as well as maternal mitochondria (and their female mtDNA) are transmitted to male offspring. Scrobicularia plana, a bivalve specie exhibiting this inheritance model possesses an important in-frame insertion of approximately 4,8 kb in its Mcox2 gene that is translated into a polypeptide of 1 892 amino acids making it the largest metazoan COX2 protein known to date. The aim of this study was to characterize the evolution and possible function of the Mcox2 insertion in S. plana through RT-PCRs, immunoassays, and bioinformatic analysis. The insertion is present amongst individuals from different populations, contains some variations in its sequence length, is rich in intrinsically disordered regions and evolves under purifying selection. The long insertion could modify the 3D structure of complex IV in the electron transport chain (ETC), impacting its function in oxidative phosphorylation (OXPHOS) which could explain low OXPHOS rates that were found in male mitochondria of DUI bivalves. The insertion could also alter male mitochondrial metabolism by interacting with other complexes of the ETC and with ATP synthase. As for other modifications of Mcox2 in DUI bivalves, a role in sex determination can also be predicted for MCOX2 in S.plana.

La protéine mitochondriale LRPPRC (leucine-rich pentatricopeptide repeat motif containing), codée par le gène nucléaire du même nom, est impliquée dans la stabilisation des ARNm mitochondriaux, particulièrement les ARNm codants pour l’assemblage du complexe IV (COX) de la chaîne respiratoire mitochondriale (OXPHOS). Le syndrome de Leigh de type canadien français (LSFC) est une maladie mitochondriale neurodégénérative caractérisée par une mutation spécifique A354V du gène Lrpprc, et par une déficience de l’activité de COX. Les organes les plus affectés sont le foie et le cerveau mais, les mécanismes associés à la progression de la maladie restent encore peu compris. Un modèle murin à délétion hépato-spécifique en LRPPRC (H-LRPPRC KO) a été créé dans le but d’étudier l’aspect hépatique du LSFC caractérisé par des dommages et une stéatose hépatique. Représentant l’objectif 1 de ce mémoire, le modèle H-LRPPRC KO a été utilisé pour une étude de caractérisation de la stéatose hépatique non-alcoolique (SHNA) sans obésité dans laquelle nous avons pu mettre en évidence une progression plus avancée de la pathologie hépatique chez les souris mâles associée à la présence d’une dysfonction cardiaque diastolique. L’objectif 2 de ce mémoire a pour but la caractérisation d’un nouveau modèle murin plus représentatif du LSFC pour ultimement trouver de nouvelles signatures/approches thérapeutiques. Nous utilisons cette fois un modèle murin développé par nos collaborateurs, à délétion inductible (KI), par le tamoxifène, de Lrpprc sur un allèle tandis que le deuxième exprime la mutation A354V spécifique au LSFC pour ainsi caractériser la maladie d’un point de vue biochimique et moléculaire. Nos premiers résultats montrent des signatures et caractéristiques comparables à celles observées chez les patient(e)s LSFC et dans le modèle H-LRPPRC KO avec une perte de poids drastique, une diminution des niveaux de la protéine LRPPRC et de COX et plusieurs perturbations du profil lipidomique dans le foie, le plasma et le cerveau. Ces résultats posent les bases biochimiques et moléculaires de ce modèle pour justifier son utilisation ultérieure pour l’évaluation des manifestations cliniques comme les atteintes musculaires et encore cognitives tel qu’observé chez les personnes atteintes de LSFC. / The mitochondrial protein LRPPRC (leucine-rich pentatricopeptide repeat motif containing), encoded by the nuclear gene of the same name, is involved in the stabilization of mitochondrial mRNAs, particularly those coding for the assembly of complex IV (COX) of the mitochondrial respiratory chain (OXPHOS). Leigh syndrome French Canadian type (LSFC) is a mitochondrial neurodegenerative disease characterized by a specific A354V mutation in the Lrpprc gene as well as a deficiency in COX activity. The most affected organs are the liver and brain, but the mechanisms associated with disease progression remain poorly understood. A hepato-specific knockout of LRPPRC mouse model (H-LRPPRC KO) was created to study the hepatic aspect of LSFC which includes liver damage and steatosis. Defined as the first objective of this master’s thesis, the H-LRPPRC KO model was used for the characterization of non-alcoholic hepatic steatosis (NAHS) without obesity in which we were able to highlight a more advanced progression of liver pathology in male mice associated with the presence of cardiac diastolic dysfunction. Furthermore, the second objective of this master’s thesis aims to characterize a new mouse model more representative of LSFC to ultimately find new therapeutic signatures/approaches. Here, we use a mouse model developed by our collaborators with tamoxifen-inducible deletion (KI) of Lrpprc on one allele, while the second one expresses the LSFC-specific A354V mutation, to characterize the disease from a biochemical and molecular perspective. Our initial results show signatures and characteristics comparable to those observed in LSFC patients as well as in the H-LRPPRC KO model, with drastic weight loss, reduced protein levels of LRPPRC and COX, and several disturbances of the lipidomic profile in liver, plasma and brain. These results lay the biochemical and molecular foundations of this model, justifying its future use in the evaluation of clinical manifestations such as muscular and cognitive impairment as observed in LSFC patients.