Kliza Katarzyna, Husnjak Koraljka
Institute of Biochemistry II, Medical Faculty, Goethe University, Frankfurt, Germany.
Front Mol Biosci. 2020 Feb 27;7:21. doi: 10.3389/fmolb.2020.00021. eCollection 2020.
Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.
泛素化通过泛素写入器（E1、E2和E3酶）、擦除器（去泛素化酶）和读取器（通过其泛素结合结构域识别泛素化蛋白质的蛋白质）的协同活性来调节几乎所有细胞过程。通过差异性地修饰细胞蛋白质组并识别这些泛素修饰，泛素化机制在空间和时间上严格调控特定细胞事件的执行。动态且复杂的泛素结构，从单泛素化、多个单泛素化、八种不同模式的同型以及众多类型的异质多聚泛素连接，实现了对细胞过程的高度动态且复杂的调控。我们讨论了研究泛素网络的现有工具和方法，包括鉴定和定量泛素修饰底物的方法，以及量化不同泛素链的长度、丰度、连接类型和结构的方法。此外，我们还总结了发现新型泛素读取器和泛素结合结构域的现有方法，以及监测和可视化泛素结合与去结合机制活性的方法。我们还讨论了现有技术的优点、缺点和局限性，以及对细胞泛素化网络进行详细时空剖析仍需要的条件。
