Department of Genetic and Evolution, Federal University of São Carlos.
Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo.
J Vis Exp. 2022 May 10(183). doi: 10.3791/63561.
Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.
泛素化是真核细胞中发生的一种翻译后修饰，对包括细胞存活、增殖和分化在内的几种生物途径的调节至关重要。它是一个可逆的过程，包括通过至少三种不同酶的级联反应将泛素共价连接到底物上，这些酶由 E1（泛素激活酶）、E2（泛素结合酶）和 E3（泛素连接酶）组成。E3 复合物在底物识别和泛素化中起着重要作用。在这里，描述了一种使用瞬时共转染编码选定底物、E3 泛素连接酶和标记泛素的质粒来评估哺乳动物细胞中底物泛素化的方案。在裂解之前，用蛋白酶体抑制剂 MG132（carbobenzoxy-leu-leu-leucinal）处理转染的细胞，以避免底物蛋白酶体降解。此外，细胞提取物进行小规模免疫沉淀 (IP) 以纯化多泛素化底物，然后使用针对泛素标签的特异性抗体进行 Western blot (WB) 检测。因此，描述了一种用于哺乳动物细胞泛素化分析的一致且简单的方案，以帮助科学家解决特定底物和 E3 泛素连接酶的泛素化问题。
