The aminopeptidase LAP3 suppression accelerates myogenic differentiation via the AKT-TFE3 pathway in C2C12 myoblasts

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

Automated summary

Skeletal muscle maintenance relies on muscle stem cells that provide myoblasts for muscle regeneration and growth. Proteasome dysfunction in skeletal muscle impairs muscle growth and development. Aminopeptidase inhibition affects the proliferation and differentiation ability of C2C12 myoblasts. Knockdown of different aminopeptidases in C2C12 myoblasts results in defective myogenic differentiation, except for leucine aminopeptidase 3 (LAP3) which promotes myogenic differentiation. Knockdown of LAP3 inhibits proteasomal proteolysis, decreases branched-chain amino acid levels, and enhances mTORC2-mediated AKT phosphorylation to promote myogenic differentiation through increased expression of myogenin.