Dataset Information

ASB2 interactome in C2C12 cells

ABSTRACT: Ubiquitination is a post-translational 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 down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2 expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant di-Gly-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 ASB2 and, therefore, potential ASB2 targets, Flag-tagged wild type ASB2, and a mutant ASB2 lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 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.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle Myoblast, Cell Culture

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD023391 | Pride | 2021-03-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180424_QEplus_BenP_Jonno_ASB2-187.raw	Raw
	20180424_QEplus_BenP_Jonno_ASB2-188.raw	Raw
	20180424_QEplus_BenP_Jonno_ASB2-189.raw	Raw
	20180424_QEplus_BenP_Jonno_ASB2-191.raw	Raw
	20180424_QEplus_BenP_Jonno_ASB2-192.raw	Raw

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Publications

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

Goodman Craig A CA Davey Jonathan R JR Hagg Adam A Parker Benjamin L BL Gregorevic Paul P

Molecular & cellular proteomics : MCP 20210129

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 ...[more]

PMID: 33516941

Similar Datasets

Project description:Ubiquitination is a post-translational modification that has been shown to have a range of effects, such as regulating protein function, protein:protein interactions, protein localization, and protein abundance by targeting proteins for degradation by the 26S proteasome. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2, is down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effect of increased ASB2 expression on skeletal muscle mass and function, we used 2-dimensional nano-ultra-high-performance mass spectrometry to investigate ASB2-induced changes to the mouse skeletal muscle proteome, and whether these were associated with changes in protein ubiquitination. The results show that in vivo recombinant adeno-associated viral vector-mediated ASB2β overexpression induces impaired intrinsic force production and progressive muscle atrophy over 12 weeks, while ASB2 knockdown induces mild muscle hypertrophy. ASB2-induced muscle atrophy and dysfunction were associated with the early down regulation of mitochondrial and contractile proteins, and the upregulation of proteins involved in proteasome-mediated protein degradation, 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 gain insights into proteins that interact with ASB2, and potential ASB2 targets, in muscle cells, flag-tagged wild type ASB2, and a mutant ASB2 with a deleted C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, we identified several novel potential ASB2β target substrates that await further investigation. Overall, this study reveals for the first time the complexity of changes to the proteome and ubiquitinome during E3 ligase-mediated skeletal muscle atrophy and dysfunction.