Hon Jason, Lu Ying
Department of Systems Biology, Harvard Medical School, Boston, MA, United States.
Department of Systems Biology, Harvard Medical School, Boston, MA, United States.
Methods Enzymol. 2019;619:225-247. doi: 10.1016/bs.mie.2018.12.031. Epub 2019 Feb 4.
The ubiquitin-proteasome system (UPS) contributes to changes in cell state and homeostatic maintenance in humans by modulating the stability of about a third of human proteins. For example, cell-cycle regulation requires a central ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts a ubiquitination cascade leading to the degradation of multiple targets. This targeted degradation is mediated by the 26S proteasome, a 2.5-MDa protein complex, which recognizes and degrades ubiquitinated proteins at rates partially controlled by the variations in ubiquitin chain topology. Substrate selectivity of ubiquitin ligases such as the APC/C and of the 26S proteasome from pools of near-identical targets reflects highly regulated kinetic mechanisms. Single-molecule techniques are powerful tools that allow distinction between differential substrate affinities and identification of reaction intermediates in complex mixtures. Here we describe fluorescence-based single-molecule imaging of in vitro ubiquitination reactions catalyzed by the APC/C and ubiquitin-dependent degradation reactions catalyzed by the 26S proteasome.
泛素 - 蛋白酶体系统（UPS）通过调节约三分之一的人类蛋白质的稳定性，对人类细胞状态的变化和稳态维持做出贡献。例如，细胞周期调控需要一种核心泛素连接酶，即后期促进复合物/细胞周期体（APC/C），它启动一个泛素化级联反应，导致多个靶标的降解。这种靶向降解由26S蛋白酶体介导，26S蛋白酶体是一种2.5兆道尔顿的蛋白质复合物，它以部分受泛素链拓扑结构变化控制的速率识别并降解泛素化蛋白质。泛素连接酶（如APC/C）和26S蛋白酶体在近乎相同的靶标库中的底物选择性反映了高度调控的动力学机制。单分子技术是强大的工具，能够区分不同的底物亲和力，并识别复杂混合物中的反应中间体。在这里，我们描述了基于荧光的单分子成像，用于观察由APC/C催化的体外泛素化反应以及由26S蛋白酶体催化的泛素依赖性降解反应。
