Davidson Gregory A, Moafian Zeinab, Sensi Amanda R, Zhuang Zhihao
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA.
Nat Protoc. 2025 Apr 25. doi: 10.1038/s41596-025-01162-8.
Protein ubiquitination, a critical regulatory mechanism and post-translational modification in eukaryotic cells, involves the formation of an isopeptide bond between ubiquitin (Ub) and targeted proteins. Despite extensive investigation into the roles played by protein ubiquitination in various cellular processes, many questions remain to be answered. A major challenge in the biochemical and biophysical characterization of protein ubiquitination, along with its associated pathways and protein players, lies in the generation of ubiquitinated proteins, either in mono- or poly-ubiquitinated forms. Enzymatic and chemical strategies have been reported to address this challenge; however, there are still unmet needs for the facile generation of ubiquitinated proteins in the quantity and homogeneity required to precisely decipher the role of various protein-specific ubiquitination events. In this protocol, we provide the ubiquitin research community with a chemical ubiquitination method enabled by an α-bromoketone-mediated ligation strategy. This method can be readily adapted to generate mono- and poly-ubiquitinated proteins of interest through a cysteine introduced to replace the target lysine, with the native cysteines mutated to serine. Using proliferating cell nuclear antigen (PCNA) as an example, we present herein a detailed protocol for generating di- and tri-Ub PCNA that contains a photo-activatable cross-linker for capturing potential reader proteins. The thioether-mediated protein ligation and purification typically takes 2-3 weeks. An important feature of our ubiquitination strategy is the ability to introduce a Michael-acceptor warhead to the linkage, allowing the generation of activity-based probes for deubiquitinases and ubiquitin-carrying enzymes such as HECT and RBR E3 ubiquitin ligases and E2 enzymes. As such, our method is highly versatile and can be readily adapted to investigate the readers and erasers of many proteins that undergo reversible ubiquitination.
蛋白质泛素化是真核细胞中一种关键的调控机制和翻译后修饰，涉及泛素（Ub）与靶蛋白之间形成异肽键。尽管对蛋白质泛素化在各种细胞过程中所起的作用进行了广泛研究，但仍有许多问题有待解答。蛋白质泛素化及其相关途径和蛋白参与者的生化和生物物理特性表征面临的一个主要挑战在于生成单泛素化或多泛素化形式的泛素化蛋白。据报道，酶促和化学策略可应对这一挑战；然而，对于以精确解读各种蛋白质特异性泛素化事件的作用所需的数量和均一性轻松生成泛素化蛋白，仍存在未满足的需求。在本方案中，我们为泛素研究群体提供了一种基于α-溴代酮介导的连接策略的化学泛素化方法。该方法可通过引入一个半胱氨酸来替代目标赖氨酸，并将天然半胱氨酸突变为丝氨酸，轻松生成感兴趣的单泛素化和多泛素化蛋白。以增殖细胞核抗原（PCNA）为例，我们在此展示了生成含有光活化交联剂以捕获潜在读取蛋白的双泛素化和三泛素化PCNA的详细方案。硫醚介导的蛋白质连接和纯化通常需要2至3周。我们泛素化策略的一个重要特点是能够在连接中引入迈克尔受体弹头，从而生成用于去泛素化酶和携带泛素的酶（如HECT和RBR E3泛素连接酶以及E2酶）的基于活性的探针。因此，我们的方法具有高度通用性，可轻松用于研究许多经历可逆泛素化的蛋白质的读取蛋白和擦除蛋白。
