Weissinger Herwig, Urschbach Moritz, Ferrari Luca, Martens Sascha, Becker Christian F W
Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, Vienna, Austria.
Max Perutz Labs, Vienna Biocenter Campus (VBC), Vienna, Austria.
J Pept Sci. 2025 Aug;31(8):e70037. doi: 10.1002/psc.70037.
Ubiquitylation is a highly conserved post-translational modification (PTM) in eukaryotes, which serves as a critical regulatory mechanism for protein homeostasis, cellular transport, signal transduction pathways, and numerous other functions. The biological function of ubiquitylation is dictated predominantly by the topology of its linkage. Deciphering ubiquitin's complex biochemistry necessitates novel synthetic methods that deliver well-defined, biosimilar ubiquitylation. To this end, a semisynthetic strategy relying on the recombinant expression of ubiquitin combined with chemoselective photocatalytic diselenide contraction (PDC) was established to enable site-selective biomimetic selenalysine-linked ubiquitylation. The modification of ubiquitin with a C-terminal selenol was fine-tuned to avoid hydrolysis. The conditions of the PDC reaction, such as solvent composition, phosphine concentration, and irradiation, were optimized for efficient ubiquitylation of a Tau F derived peptide. Furthermore, it was demonstrated that the selenalysine linkage undergoes efficient cleavage by deubiquitylating enzymes, comparable to the native isopeptide linkage. The presented method expands the toolbox of site-selective ubiquitylation techniques. It is tolerant to many functional groups and will help to further elucidate the complexities of ubiquitylation.
泛素化是真核生物中一种高度保守的翻译后修饰（PTM），它作为蛋白质稳态、细胞运输、信号转导途径及许多其他功能的关键调节机制。泛素化的生物学功能主要由其连接拓扑结构决定。破解泛素复杂的生物化学特性需要新的合成方法来实现明确的、生物相似的泛素化。为此，建立了一种依赖泛素重组表达并结合化学选择性光催化二硒化物缩合（PDC）的半合成策略，以实现位点选择性的仿生硒赖氨酸连接的泛素化。对带有C端硒醇的泛素修饰进行了微调以避免水解。对PDC反应条件，如溶剂组成、膦浓度和辐照等进行了优化，以实现Tau F衍生肽的高效泛素化。此外，还证明了硒赖氨酸连接可被去泛素化酶有效切割，与天然异肽键连接相当。所提出的方法扩展了位点选择性泛素化技术的工具库。它对许多官能团具有耐受性，并将有助于进一步阐明泛素化的复杂性。
