X-linked Duchenne muscular dystrophy is caused by primary abnormalities in the Dmd gene causing the almost complete absence of the membrane cytoskeletal protein dystrophin in contractile tissues. Exon skipping promises the conversion of the severe Duchenne phenotype into the more benign Becker’s form of this neuromuscular disease. In this respect, superior sets of diagnostic tools are needed for the proper evaluation of a successful transformation of dystrophinopathies. In order to establish a comprehensive protein marker signature of muscular dystrophy, we plan to study the dystrophin complexome by interaction and expression proteomics. Since dystrophin and its associated glycoprotein complex is a highly dynamic assembly, its biochemical isolation and the subsequent categorization of low abundant binding partners is not trivial. However, the stabilization of relatively weak interactions between proteins that exhibit greatly differing physicochemical properties can be achieved by chemical crosslinking. Here, we plan to combine chemical crosslinking and co-immunoprecipitation of the native dystrophin network with mass spectrometry for the comparative proteomic profiling of dystrophic muscle tissues. The underlying objective of this proposal is to increase our biochemical understanding of the complexity of the dystrophin network and the secondary effects of dystrophin deficiency in X-linked muscular dystrophy. The main scientific goals of this project are (i) the isolation of the chemically crosslinked/immunoprecipitated dystrophin complex, (ii) the immunochemical analysis of the dystrophin-glycoprotein complex and its interactions with the basal lamina, sarcolemma and actin cytoskeleton, (iii) the mass spectrometric identification of proteins associated with the dystrophin network, and (iv) the determination of secondary effects in dystrophin-deficient tissues ranging from moderately affected muscles to the severely necrotic and fibrotic diaphragm from the mdx mouse model of Duchenne muscular dystrophy. A better comprehension of the molecular pathogenesis of dystrophinopathy may be helpful to improve the identification of novel therapeutic targets, diagnostics and therapy monitoring.
