Krist David T, Foote Peter K, Statsyuk Alexander V
Northwestern University, Department of Chemistry, Chemistry of Life Processes Institute, Evanston, Illinois.
Curr Protoc Chem Biol. 2017 Mar 2;9(1):11-37. doi: 10.1002/cpch.17.
HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3∼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley & Sons, Inc.
HECT E3泛素连接酶（已知约有28种）与真核生物中的许多表型相关，是重要的药物靶点。然而，用于筛选HECT E3小分子抑制剂的检测方法很复杂，需要ATP、泛素（Ub）、E1、E2和HECT E3酶，会产生三种共价硫酯酶中间体E1∼Ub、E2∼Ub和HECT E3∼Ub（其中∼表示硫酯键）以及多聚泛素链混合物。为了降低检测的复杂性，我们开发了一类新型荧光探针UbFluor，它可作为与HECT E3机制相关的假底物。这些探针与HECT E3的催化半胱氨酸发生直接转硫醇反应，产生具有催化活性的HECT E3∼Ub硫酯并伴随荧光团释放。因此，荧光偏振检测可以持续监测HECT E3对UbFluor的消耗，以及由生化点突变或HECT E3活性的小分子调节导致的UbFluor消耗变化。© 2017年约翰威立父子出版公司
