G.P.7.02 Studying the functional pathogenesis of nebulin: A giant myofibrillar protein

Nemaline myopathy (NM) is a neuromuscular disorder characterized by the presence of nemaline (rod-like) bodies in patient muscles. Defects in the nebulin gene (NEB) and skeletal muscle α-actin (ACTA1) are the most common causes of NM. The 249 kb NEB gene comprising 183 exons remains one of the least understood molecules of striated muscle. Nebulin is a large (600–900 kDa) protein found in vertebrate skeletal muscles. It is believed to dictate actin thin filament length in skeletal muscle and is thought to have many other roles in the sarcomere, but functional studies are required to confirm this hypothesis. The core objective of this study is to help elucidate the functional pathogenesis of NM and related disorders caused by mutations in NEB. The exons selected in the studies include the Ashkenazi founder mutation (exon 55 deletion) and Finnish founder mutations (exon 122, 151) along with other exons (exon 54, 78, exon 143 and 144). PCR, in-vitromutagenesis, yeast two-hybrid assays, actin and tropomyosin binding assays and phosphorylation assays will be employed to achieve our goals in current studies. We have cloned all the wild type and mutated constructs for the pathogenesis studies including exons 54, 55, 78, 122 and 151. After optimizing the protein production from all these constructs, we will proceed with F-actin and tropomyosin binding assays. For yeast two-hybrid studies exons 143/144 have also been successfully cloned. The initial results on toxicity assays show that the exogenous clones are non-toxic to the yeast host cells. Screening assays and the interaction studies are in progress. The nebulin functional studies employed herein have not been performed earlier.