Vela-Rodríguez Carlos, Scarpulla Ilaria, Ashok Yashwanth, Lehtiö Lari
Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Finland.
Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Finland.
SLAS Discov. 2023 Dec;28(8):365-375. doi: 10.1016/j.slasd.2023.08.005. Epub 2023 Aug 12.
Ubiquitination is a reversible protein post-translational modification in which consequent enzymatic activity results in the covalent linking of ubiquitin to a target protein. Once ubiquitinated, a protein can undergo multiple rounds of ubiquitination on multiple sites or form poly-ubiquitin chains. Ubiquitination regulates various cellular processes, and dysregulation of ubiquitination has been associated with more than one type of cancer. Therefore, efforts have been carried out to identify modulators of the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor ubiquitination activity of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a good signal window with a robust average Z' factor of 0.76 on 384-well microplates, indicating a good assay for screening inhibitors in a high-throughput setting. From a validatory screening experiment, we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. We also demonstrate that the method can be expanded to study deubiquitinases, such as USP28, that reduce FRET due to hydrolysis of fluorescent poly-ubiquitin chains.
泛素化是一种可逆的蛋白质翻译后修饰，其后续的酶促活性导致泛素与靶蛋白共价连接。一旦被泛素化，蛋白质可以在多个位点经历多轮泛素化或形成多聚泛素链。泛素化调节各种细胞过程，而泛素化失调与不止一种类型的癌症相关。因此，人们一直在努力寻找泛素化级联反应的调节剂。在此，我们展示了一种基于荧光共振能量转移（FRET）的检测方法的开发，该方法使我们能够监测RING型E3泛素连接酶DTX3L的泛素化活性。我们的方法在384孔微孔板上显示出良好的信号窗口，平均Z'因子稳健，为0.76，表明该方法适合在高通量环境下筛选抑制剂。通过验证性筛选实验，我们鉴定出了首批在低微摩尔范围内抑制DTX3L的分子。我们还证明，该方法可以扩展到研究去泛素化酶，如USP28，其通过水解荧光多聚泛素链降低FRET。
