Research of ATAD3 partners and study of the effect of invalidation on the adipocyte differentiation.
ATAD3 is a vital inner membrane mitochondrial ATPase with unknown function but widely expressed in animals and plants. The main project was to study the role of ATAD3 in a cellular model of differentiation, the one used being adipocyte-like 3T3-L1 cells induced by insuline. Induction of these cells provokes the start of the adipogenesis program which precedes lipid droplets synthesis (lipogenesis). We observed that along differentiation, a synthesis and a remodelling of mitochondria occur. ATAD3, which is expressed in pre-adipocytes, is increased before lipid droplets accumulation and before mitochondrial synthesis and remodelling and is maintained all along differntiation. We have generated cell lines with constitutive invalidation of ATAD3 (siRNA) and studied the differentiation of these cells. We have shown that ATAD3 invalidation inhibits lipogenesis and mitochondrial remodelling without any inhibition of insulin transduction signal neither inhibition of mitochondrial protein synthesis. The phenotype was rescued by ATAD3 transfection and complemented by over-expression of Drp1. These results show that ATAD3 is a necessary and limiting factor in adypocyte differentiation and lipogenesis. Three parallel projects completed this thesis work: The first was to contribute to purification of recombinant human ATAD3 produced in yeast. These experiments didn't allow us to reveal the ATPase activity of ATAD3 but allowed us to obtain a significant quantity of purified protein that will be useful to generate antibodies and also for others applications. The second was to study ATAD3 isoforms expression profiles in human and rodent samples. These experiments allowed the description of several isoforms of ATAD3 with tissue-specific expression. A third project was the identification of ATAD3 protein partners in order to try to understand its function. Using labelled ATAD3 protein probes (in vitro transcription/traduction), we performed interaction tests by Far Western. These experiments led to the identification of seve