Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan.
Protein Sci. 2023 Oct;32(10):e4766. doi: 10.1002/pro.4766.
Ubiquitin (Ub)-conjugating enzymes (E2s) transfer activated Ub from Ub-activating enzymes (E1s) to substrates and are associated with various cancers and neurological disorders. In this study, the unique properties of E2-binding and auto-ubiquitination of artificial RING fingers (ARFs) were demonstrated in ubiquitination assays. Circular dichroism spectra indicated the characteristic structures of ARFs. Point mutations of PKLTC in ARF by tryptophan (Trp) resulted in dramatic changes in E2 specificity and the type of Ub chain elongation of mono- and polyubiquitination. The Trp residue was a cue that changed the ubiquitination activity of ARF via E2-binding. Furthermore, the ARF mutants interacted with all 11 E2s and then promoted auto-ubiquitination. Thus, the use of the ARF mutants allowed specific detection of E2 activities during ubiquitination. The present study opens up a new avenue for researching E2 activities related to the fatal diseases.
泛素（Ub）连接酶（E2s）将活化的 Ub 从 Ub 激活酶（E1s）转移到底物上，与各种癌症和神经紊乱有关。在这项研究中，通过泛素化测定，证明了人工 RING 指（ARF）的 E2 结合和自身泛素化的独特性质。圆二色性光谱表明了 ARF 的特征结构。通过色氨酸（Trp）对 ARF 中 PKLTC 的点突变导致 E2 特异性和单泛素化和多泛素化的 Ub 链延伸类型发生剧烈变化。色氨酸残基是一个线索，通过 E2 结合改变了 ARF 的泛素化活性。此外，ARF 突变体与所有 11 种 E2 相互作用，然后促进自身泛素化。因此，使用 ARF 突变体可以在泛素化过程中特异性检测 E2 活性。本研究为研究与致命疾病相关的 E2 活性开辟了新途径。
