Department of Physiology, Centre for Muscle Research (CMR), The University of Melbourne, Victoria, Australia; Australian Institute for Musculoskeletal Science (AIMSS), Sunshine Hospital, The University of Melbourne, St Albans, Victoria, Australia.
Department of Physiology, Centre for Muscle Research (CMR), The University of Melbourne, Victoria, Australia; Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia.
Mol Cell Proteomics. 2021;20:100050. doi: 10.1016/j.mcpro.2021.100050. Epub 2021 Jan 29.
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.
泛素化是一种翻译后蛋白质修饰，已被证明具有多种效应，包括调节蛋白质功能、相互作用、定位和降解。我们之前已经表明，肌肉特异性泛素 E3 连接酶 ASB2β 在肌肉生长模型中下调，并且过表达 ASB2β 足以诱导肌肉萎缩。为了深入了解增加 ASB2β 表达对骨骼肌质量和功能的影响，我们使用液相色谱串联质谱法通过对残留双甘肽修饰肽的平行分析，研究了 ASB2β 介导的骨骼肌蛋白质组和泛素组的变化。结果表明，病毒载体介导的 ASB2β 在鼠肌肉中的过表达导致进行性肌肉萎缩和产生力的能力受损，而 ASB2β 敲低诱导轻度肌肉肥大。ASB2β 诱导的肌肉萎缩和功能障碍与线粒体和收缩蛋白丰度的早期下调以及参与蛋白酶体介导的蛋白质降解（包括其他 E3 连接酶）、蛋白质合成和细胞骨架/肌节的蛋白质上调有关。ASB2β 的过表达还导致蛋白质泛素化的明显变化；然而，泛素化状态的变化与蛋白质丰度之间没有简单的关系。为了研究与 ASB2β 相互作用的蛋白质，因此是潜在的 ASB2β 靶标，我们从 C2C12 肌管中免疫沉淀了带有 Flag 标签的野生型 ASB2β 和缺乏 C 末端 SOCS 盒结构域的突变体 ASB2β（dSOCS），并进行了无标记蛋白质组分析，以确定 ASB2β 相互作用组。发现 ASB2β 与一系列细胞骨架和核蛋白相互作用。当与体内泛素组学数据结合时，我们的研究鉴定了新型潜在的 ASB2β 靶标底物，值得进一步研究。这些发现为 E3 连接酶介导的骨骼肌萎缩和功能障碍过程中发生的蛋白质组和泛素组变化的复杂性提供了新的见解。
