Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2β

• Published in: Molecular & cellular proteomics Vol. 20; p. 100050
• Main Authors: Goodman, Craig A., Davey, Jonathan R., Hagg, Adam, Parker, Benjamin L., Gregorevic, Paul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021; American Society for Biochemistry and Molecular Biology
• Subjects: adeno-associated viral vectors; Animals; autophagy; Cell Line; Female; filamin; Male; Mice; Mice, Inbred C57BL; mitochondria; muscle contraction; Muscle Proteins - metabolism; Muscle, Skeletal - metabolism; Muscle, Skeletal - pathology; Muscular Atrophy - metabolism; proteasome; protein degradation; Proteome; skeletal muscle atrophy; titin; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; muscle contraction; proteasome; skeletal muscle atrophy; EDL; TFA; titin; CSA; DUB; GFP; filamin; dSOCS; UPS; diGly; WT; autophagy; mitochondria; GO; SOCS; 2D nano-UHPL-MS/MS; CMV; PVDF; TA; OMM; rAAV; TCA; ubiquitination; ETC; TEAB; ROS; adeno-associated viral vectors; UIM; E-C; TMT; HBSS; MCS; mTORC1; protein degradation
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2021.100050

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2β, is downregulated in models of muscle growth and that overexpression ASB2β is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2β expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2β-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2β overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2β knockdown induces mild muscle hypertrophy. ASB2β-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2β and, therefore, potential ASB2β targets, Flag-tagged wild-type ASB2β, and a mutant ASB2β lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2β interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2β target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction. [Display omitted] •Proteomic and ubiquitinomic analysis of increased ASB2β in skeletal muscle•ASB2β increased the abundance of proteome and cytoskeletal proteins•ASB2β reduced the abundance of mitochondrial and contractile proteins•No simple relationship between changes in protein abundance and ubiquitination The E3 ubiquitin ligase ASB2β has been identified as a regulator of skeletal muscle mass. To gain insights into potential mechanisms of action, mouse muscles expressing a Flag-tagged ASB2β were investigated using quantitative proteomic methods. The results identified ASB2β-induced changes in the abundance and ubiquitination of proteins associated with mitochondria, the sarcomere, and the cytoskeleton. Additional in vitro studies identified novel putative ASB2β target substrates. The results highlight the complex relationship between protein abundance and ubiquitination in ASB2β-mediated muscle adaptation.