Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ

Abstract

Inflammation in muscle induces the synthesis of mediators that can impair protein synthesis and enhance proteolysis, and when sustained lead to muscle atrophy. Furthermore, muscle-derived mediators that are secreted may participate in disrupting the function of other peripheral organs. Selective identification of newly synthesized proteins can provide insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those proteins that are up- or down-regulated in response to inflammation. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from their exposure to the inflammatory mediators lipopolysaccharide (LPS) and interferon (IFN)-γ for either a short (4 h) or prolonged (16 h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1523 proteins and report their detection from three independent samples of each condition at each time point. The identified nascent proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these proteins was identified only in treated samples and has functional characteristics consistent with the synthesis of specific new proteins in response to LPS/IFNγ. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of new synthesis of proteins in both protein homeostasis and in proteome responses to stimuli in C2C12 myotubes. Our results reveal a profile of actively translating proteins for specific cellular components and biological processes in normal C2C12 myotubes and a different enrichment of proteins in response to LPS/IFNγ. Collectively, our data disclose a highly interconnected network that integrates the regulation of cellular proteostasis and reveal a diverse immune response to inflammation in muscle which may underlie the concomitantly observed atrophy and be important in inter-organ communication.

Keywords: Immune response; Inflammation; LPS; Muscle; Proteome.