Blazev Ronnie, Zee Barry M, Peckham Hayley, Ng Yaan-Kit, Lewis Christopher T A, Zhang Chengxin, McNamara James W, Goodman Craig A, Gregorevic Paul, Ochala Julien, Steyn Frederik J, Ngo Shyuan T, Stokes Matthew P, Parker Benjamin L
Department of Anatomy and Physiology, The University of Melbourne, Parkville, VIC 3010, Australia; Centre for Muscle Research, The University of Melbourne, Parkville, VIC 3010, Australia.
Cell Signaling Technology, Danvers, MA 01923, USA.
Cell Rep Methods. 2025 May 19;5(5):101048. doi: 10.1016/j.crmeth.2025.101048. Epub 2025 May 9.
UFMylation is a ubiquitin-like protein modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins and regulates several cellular processes such as protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate remnant UFMylated peptides and identification by mass spectrometry. We used this approach to identify >200 UFMylation sites from various mouse tissues, revealing extensive modification in skeletal muscle. In vivo knockdown of the E2 ligase, UFC1, followed by enrichment and analysis of remnant UFMylated peptides quantified concomitant down-regulation and validation of a subset of modification sites, particularly myosin UFMylation. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with amyotrophic lateral sclerosis (plwALS). Quantification of UFMylation sites in these biopsies with multiplexed isotopic labeling reveal prominent increases in myosin UFMylation. Our data suggest that in vivo UFMylation is more complex than previously thought.
泛素样修饰因子1（UFM1）化是一种将泛素折叠修饰因子1（UFM1）应用于底物蛋白的类泛素蛋白修饰，可调节多种细胞过程，如蛋白质质量控制。在此，我们描述了一种基于抗体的富集方法的开发，该方法用于免疫沉淀残余的泛素样修饰因子1（UFM1）化肽段，并通过质谱进行鉴定。我们使用这种方法从各种小鼠组织中鉴定出>200个泛素样修饰因子1（UFM1）化位点，揭示了骨骼肌中的广泛修饰。在体内敲低E2连接酶UFC1，随后对残余的泛素样修饰因子1（UFM1）化肽段进行富集和分析，定量了伴随的下调情况，并验证了一部分修饰位点，特别是肌球蛋白的泛素样修饰因子1（UFM1）化。此外，我们表明，在肌萎缩侧索硬化症患者（plwALS）的骨骼肌活检中，泛素样修饰因子1（UFM1）化增加。用多重同位素标记对这些活检中的泛素样修饰因子1（UFM1）化位点进行定量分析，发现肌球蛋白的泛素样修饰因子1（UFM1）化显著增加。我们的数据表明，体内泛素样修饰因子1（UFM1）化比以前认为的更为复杂。
