Dataset Information

ASB2beta-induced changes to the skeletal muscle proteome and ubiquitinome

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle, Skeletal Muscle Cell Line

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD020040 | Pride | 2021-03-18

REPOSITORIES: Pride

ACCESS DATA

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 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.

Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

Dataset Information

ASB2beta-induced changes to the skeletal muscle proteome and ubiquitinome

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure