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    Friday Séminaire 17/03/2017

    Friday Séminaire 17/03/2017

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    Séminaire de Anna Campalans

    Invitée par Arnaud Chevrollier

    Le 17 mars 2017

    Un séminaire sera donné par le Anna Campalans, Institut de Radiobiologie Cellulaire et Moléculaire, CEA, Fontenay aux Roses, France

    «A critical role of the DNA repair protein OGG1 inmitochondrial physiology»

    Les étudiants de médecine/pharmacie devant valider certains modules (M1 et M2) devront impérativement venir avec leur fiche personnelle de validation des séminaires qui sera alors tamponnée.

    12h00 - 13h00
    ICO site Paul Papin, amphithéâtre

     secr-inserm-angers @

        Accumulation of mitochondrial DNA damage has been associated with the development of pathologies such as cancer and neurodegenerative disorders (Parkinson and Alzheimer diseases). Mitochondria host the electron transport chain and they are one of the main sources of ROS in the cells. Mitochondrial DNA (mtDNA), localised in close proximity to the inner mitochondrial membrane is thus highly exposed to oxidative damage. One of the most frequent alterations induced by ROS is a product of oxidation of guanine, 8-oxoG, that is repaired by the Base excision repair (BER) pathway. BER of 8-oxoG is initiated both in nuclei and in mitochondria by the DNA glycosylase activity of OGG1. The downstream BER proteins required to finish the repair (an AP endonuclease, a polymerase and a ligase) have all been detected in mitochondria and are all encoded by the nuclear genome. In human cells, up to 8 different isoforms of OGG1 can be potentially generated from the same gene by alternative splicing. All the isoforms share their Nterminal region harbouring a canonical Mitochondrial Targeting Sequence (MTS). Only the alpha-OGG1 isoform has a Nuclear Localisation Signal (NLS) at its C-terminus and has been considered to be the enzyme present in the nucleus. Which isoform of OGG1 is responsible for the glycosylase activity in the mitochondria is still an open question. The beta-OGG1, considered for a long time to be the mitochondrial isoform has been shown to lack the DNA glycosylase activity. The aim of our project was to determine whether the alpha-OGG1 could also be localized into the mitochondria and could play a role in the maintenance of mitochondrial DNA. Using a combination of biochemical and imaging techniques, we demonstrate that the alpha-OGG1 is indeed present in both nucleus and mitochondria and that nuclear and mitochondrial targeting signals are both functional and essential to determine the subcellular localisation of the enzyme. Confocal and super-resolution microscopy were used to elucidate the sub-mitochondrial localisation of the enzyme and clearly show the association of alpha-OGG1 to the mtDNA in mitochondrial nucleoids.
        Several studies have reported a mitochondrial dysfunction in cells isolated from OGG1 knock-out mice after exposure to oxidative stress. In order to probe the role of alpha-OGG1 in mitochondria, we transfected human U2OS cells with a siRNA against OGG1 and exposed them to the oxidizing agent menadione. We observed a higher mitochondrial ROS production, a loss of mitochondrial membrane potential and increases in mitochondrial fragmentation and mitophagy. These mitochondrial dysfunctions were complemented by the WT version of alpha-OGG1 but not by the expression of an isoform that is deficient in its mitochondrial import (OGG1-deltaMTS), indicating that the presence of the protein in the mitochondria is essential for the protective effect. To determine if the glycosylase activity of alpha-OGG1 is required for the maintenance of mitochondrial physiology under oxidative stress, we introduced the point mutation K249Q in the OGG1 sequence that results in the loss of the enzymatic activity. While the OGG1(K249Q) mutant protein shows the same subcellular localization than the WT, it could not complement the mitochondrial phenotypes of the OGG1 depleted cells. Taken together, our results indicate that the DNA glysosylase activity of alpha-OGG1 in mitochondria is required to protect the cells from oxidative stress. The mitochondrial dysfunctions induced by oxidative stress in OGG1 deficient cells are observed from half an hour after the end of the treatment. It is thus difficult to imagine that they are the consequence of the accumulation of mutations in mtDNA. We will discuss the possible molecular mechanisms underlying the mitochondrial phenotypes in OGG1-deficient cells.