Liu Yaoqi, Dong Chaoqing, Ren Jicun
School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China.
ACS Omega. 2023 Sep 22;8(39):36588-36596. doi: 10.1021/acsomega.3c06078. eCollection 2023 Oct 3.
Deubiquitination is a reverse post-translational modification of ubiquitination and plays significant roles in various signal transduction cascades and protein stability. The p53 is a very important tumor-suppressor protein and closely implicates more than 50% of human cancers. Although extracellular studies on the deubiquitination of p53 were reported, the process of p53 deubiquitination in living cells due to the shortage of an efficient in situ method for single living cells is still not clear. In this study, we described an in situ method for studying p53 deubiquitination in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. We first constructed the stable cell line expressing EGFP-Ub-p53-mCherry as the substrate of p53 deubiquitination. Then, we established a method for in situ monitoring of the deubiquitination of p53 in living cells. Based on the amplitudes of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy curves from living cells, we obtained the deubiquitination percentage for evaluating the level of p53 protein deubiquitination. Furthermore, we studied the effects of ubiquitin structures on p53 deubiquitination in living cells and found that the C-terminal Gly75-Gly76 motif of ubiquitin is a key location for p53 deubiquitination and the deubiquitination cannot occur when ubiquitin lacks the C-terminal Gly75-Gly76 motif. Our results documented that the developed strategy is an efficient method for in situ study of deubiquitination of proteins in living cells.
去泛素化是泛素化的一种反向翻译后修饰，在各种信号转导级联反应和蛋白质稳定性中发挥着重要作用。p53是一种非常重要的肿瘤抑制蛋白，与超过50%的人类癌症密切相关。尽管已经报道了关于p53去泛素化的细胞外研究，但由于缺乏一种针对单个活细胞的高效原位方法，p53在活细胞中的去泛素化过程仍不清楚。在本研究中，我们描述了一种通过将荧光互相关光谱与荧光蛋白标记技术相结合来研究活细胞中p53去泛素化的原位方法。我们首先构建了表达EGFP-Ub-p53-mCherry的稳定细胞系作为p53去泛素化的底物。然后，我们建立了一种原位监测活细胞中p53去泛素化的方法。基于活细胞的荧光相关光谱和荧光互相关光谱曲线的振幅，我们获得了去泛素化百分比，用于评估p53蛋白去泛素化的水平。此外，我们研究了泛素结构对活细胞中p53去泛素化的影响，发现泛素的C末端Gly75-Gly76基序是p53去泛素化的关键位置，当泛素缺乏C末端Gly75-Gly76基序时，去泛素化不能发生。我们的结果证明，所开发的策略是一种在活细胞中原位研究蛋白质去泛素化的有效方法。
