A.O.7 - Research for the pathogenesis and therapy of dysferlinopathy using proteomics approach

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Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in dysferlin gene (DYSF). There are two main phenotypes including Miyoshi muscular dystrophy and limb-girdle muscular dystrophy type 2B. DYSF is composed of 55 exons coding for 2,080 amino acid residues. DYSF is expressed in the plasma membrane of skeletal muscles and is involved in calcium-mediated membrane fusion events and membrane repair. We previously identified over 50 different mutations across the entire DYSF in approximately 60% of the patients. Our research theme is focusing on these DYSF and DYSF-related proteins. Recent advances have identified some components of the cell membrane repair process, particularly those involved in a putative pathway specific to striated muscles. Two important members are DYSF and Mitsugumin 53 (MG53). Compared to DYSF, molecular size of MG53 is rather small and can be expressed and work as membrane repair molecules. To find the binding partner of DYSF, which can be working as the similar functional role, would be a good clue to find the therapeutic strategy of dysferlinopathy. Using recombinant proteins and affinity column binding methods combined with LC/MS/MS, we identified novel DYSF-binding proteins. Protein X, one of the novel proteins, is considered to be an essential component of the membrane repair process. To directly evaluate the membrane repair capacity of the X knockdown C2C12 cells, we measured FM fluorescent dye entry after laser-induced damage to cells. The wild-type cells could effectively reseal sarcolemmal membranes, as they showed only minimal FM dye entry after laser damage. In contrast, significant entry of FM dye into the X knockdown cells was observed after laser-induced damage, suggesting insufficient membrane repair in X-deficient cells. To elucidate the molecular function of protein X in dysferlinopathy condition, we will also use several kinds of myoblasts originated from dysferlinopathy patients.