Department of Chemistry, Tufts University, Medford, MA 02155, USA.
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
Angew Chem Int Ed Engl. 2024 Mar 22;63(13):e202319579. doi: 10.1002/anie.202319579. Epub 2024 Feb 19.
A cascade of three enzymes, E1-E2-E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method-targeted charging of ubiquitin to E2 (tCUbE)-that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2-E3-substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS.
级联的三种酶，E1-E2-E3，负责将泛素转移到靶蛋白上，从而控制细胞信号转导的许多不同方面。尽管 E2 影响底物的身份、链的多样性和拓扑结构，但它的作用在很大程度上被忽视了。在这里，我们报告了一种针对泛素转移酶 E2 的方法（tCUbE），它可以在活哺乳动物细胞中追踪标记的泛素通过其整个酶级联。我们利用这种方法来揭示新的靶标，其泛素化依赖于 UbcH5a E2 活性。我们证明，tCUbE 可以广泛应用于多种 E2 和不同的人类细胞系。tCUbE 非常适合于研究感兴趣的 E2-E3-底物级联，或者将以前未识别的级联拼接在一起，从而阐明 UPS 的整个分支，并提供有价值的见解，这对于确定 UPS 中的新治疗靶点将非常有用。
