MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.74 Studies on a potential interaction between nebulin alternative isoform S21b and ferritin light chain (FTL)

Abstract

Nebulin (600-900 kDa) is a giant filamentous protein located in the thin filament of the skeletal muscle sarcomere. It has a highly modular structure, consisting of more than 200 actin-binding simple repeats along the length of the protein. The simple repeats in the central part of the protein are further organised into super repeats (numbered S1-S22). Mutations in the nebulin gene (NEB, 183 exons) are the most common cause of recessively inherited nemaline myopathy. Due to extensive alternative splicing, hundreds of different nebulin isoforms are expressed in skeletal muscle. Nebulin super repeat 21 (S21) is located close to the Z disc-associated part of the protein. The mutually exclusive exons 143 and 144 give rise to two alternative isoforms, S21a and S21b, respectively. These alternative super repeats differ in both charge and hydrophobicity. Our recent studies have demonstrated that the first isoform expressed during muscle development is S21b. Later, a regulated expression of S21a begins, and is often strong in fast myofibres, while S21b expression remains relatively uniform across different fibre types. Despite clear differences in structure and expression, the exact function of these distinct nebulin isoforms remains to be elucidated. To identify potential interaction partners of S21a and S21b, we performed yeast-two-hybrid experiments. The results indicated that only one of the alternative isoforms, S21b, binds to the light chain subunit of the iron-storage protein ferritin (FTL). The ubiquitously expressed FTL is responsible for iron availability in the cell. The aim of our present study is to confirm the interaction between S21b and FTL by two methods: microscale thermophoresis (MST) and GST-pull-down. The results will form a basis for understanding the functional differences between the alternative super repeats S21a and S21b. The results, if confirmed, would suggest a novel role of nebulin as a regulator of iron storage in skeletal muscle.