Studying the pathogenesis of nebulin-caused nemaline myopathy and related disorders

Nebulin is a giant (600–900 kDa) thin filament actin-binding protein that regulates thin filament length during sarcomere assembly in skeletal muscle. Mutations in the nebulin gene (NEB) cause the autosomal recessive muscle disorder nemaline myopathy (NM). NM is a neuromuscular disorder characterized by the presence of nemaline (rod-like) bodies in patient muscles. Defects in the NEB and skeletal muscle α -actin (ACTA1) genes are the most common causes of NM. The 249 kb NEB gene comprising 183 exons remains one of the most challenging molecules of striated muscle. Majority of nebulin consists of 30–35 amino acid long repetitive modules called simple repeats. Most of the simple repeats are arranged into seven-module super repeats. Each simple repeat has a predicted alpha-helical secondary structure and an SDXXYK motif that serves as actin-binding site. A second motif, WLKGIGW, is present once in each super repeat, and is thought to serve as a tropomyosin-binding site. We have initiated functional studies of nebulin in which yeast two-hybrid assays, actin and tropomyosin binding assays and phosphorylation assays will be employed. The exons selected for the studies include Ashkenazi founder mutation (exon 55 deletion) and Finnish founder mutations (exon 122, 151) along with other exons (exon 54, 78, exon 143 and 144). After successful cloning for the pathogenetic studies of exons 54, 55, 78, 122 and 151, protein production has been optimized and F-actin binding assays have been performed. To better understand the physiological roles of exon 143/144 isoforms, we have screened the human skeletal Matchmaker cDNA library for new binding partners by using the yeast two-hybrid system with exons 143/144 as the bait. Interaction studies have revealed interesting candidate partners binding to the regions in question. The core objective of this ongoing study is to help elucidate the functional pathogenesis of NM and related disorders.