Résumé :
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To improve our understanding of the molecular pathways involved in the pathogenesis of muscular dystrophies and to allow identification of specific processes that may lead to future therapeutic strategies, we chose a domain-based yeast-two hybrid (Y2H) approach to establish a large scale protein-protein interaction (PPI) map of the skeletal muscle cell centred on proteins involved in LGMDs. An initial set of 13 proteins involved in recessive LGMD and/or in muscular atrophic processes were used as baits to perform high-throughput Y2H screenings of a muscular cDNA prey library. The resulting prey interacting proteins were analysed to select a series of new baits for additional screenings. This process was repeated twice and lead to three rounds of screenings. The entire project consisted in 87 high-throughput Y2H screenings and identified more than 1100 proteins that constitute a global network of more than 1600 PPI, with an average of 21 and 1.4 connections per bait and prey, respectively. For each PPI, a statistical score was computed to predict its reliability and coordinates of the prey interacting domains are available. Following the achievement of this muscular interactome map, our Y2H results were compared to public PPI databases and a validation process was initiated to experimentally confirm a subset of interactions by two complementary techniques: in vitro bimolecular assays (Homogeneous Time-Resolved Fluorescence) and co-immunoprecipitation experiments. Finally, comparison of the current list of known neuromuscular disorder genes with our interactome revealed that 30% of them were connected in our map. The availability of the muscular interactome data will undoubtedly expand our knowledge of the function of the proteins involved in neuromuscular disorders and will permit us to go one step further towards the dynamic and systemic comprehension of the muscle system and towards the understanding of the pathophysiology of the diseases.
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