School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
Analyst. 2023 Aug 7;148(16):3768-3775. doi: 10.1039/d3an00890h.
Newly synthesized proteins are closely related to a series of biological processes, including cell growth, differentiation, and signaling. The post-translational modifications (PTMs) of newly synthesized proteins help maintain normal cellular functions. Ubiquitination is one of the PTMs and plays a prominent role in regulating cellular functions. Although great progress has been made in studying the ubiquitination of newly synthesized proteins, the monitoring of the ubiquitination of newly synthesized proteins in living cells still remains challenging. In this study, we propose a new method for measuring the ubiquitination of newly synthesized proteins in living cells by combining a click reaction with fluorescence cross-correlation spectroscopy (FCCS). In this study, a puromycin derivative (Puro-TCO) and a fluorescence probe (Bodipy-TR-Tz) were synthesized, and then, the newly synthesized proteins in living cells were labelled with Bodipy-TR the click reaction between Puro-TCO and Tz. Ubiquitin (Ub) in living cells was labelled with the enhanced green fluorescence protein (EGFP) by fusion using a gene engineering technique. FCCS was used to quantify the newly synthesized proteins with two labels (EGFP and Bodipy-TR) in living cells. After measurements, the cross-correlation (CC) value was used to evaluate the ubiquitination degree of proteins. Herein, we established a method for monitoring the ubiquitination of newly synthesized proteins with EGFP-Ub in living cells and studied the effects of the ubiquitin E1 enzyme inhibitor on newly synthesized proteins. Our preliminary results document that the combination of FCCS with a click reaction is an efficient strategy for studying the ubiquitination of newly synthesized proteins in living cells. This new method can be applied to basic research in protein ubiquitination and drug screening at the living-cell level.
新合成的蛋白质与一系列生物过程密切相关，包括细胞生长、分化和信号转导。新合成蛋白质的翻译后修饰（PTMs）有助于维持正常的细胞功能。泛素化是一种 PTM，在调节细胞功能方面起着重要作用。尽管在研究新合成蛋白质的泛素化方面已经取得了很大进展，但在活细胞中监测新合成蛋白质的泛素化仍然具有挑战性。在这项研究中，我们提出了一种新的方法，通过结合点击反应和荧光相关光谱（FCCS）来测量活细胞中新合成蛋白质的泛素化。在这项研究中，合成了一个嘌呤霉素衍生物（Puro-TCO）和一个荧光探针（Bodipy-TR-Tz），然后用 Bodipy-TR 标记活细胞中的新合成蛋白质，通过点击反应与 Puro-TCO 结合。使用基因工程技术，将泛素（Ub）融合到增强型绿色荧光蛋白（EGFP）上进行标记。使用 FCCS 来量化活细胞中带有两个标签（EGFP 和 Bodipy-TR）的新合成蛋白质。测量后，使用交叉相关（CC）值来评估蛋白质的泛素化程度。在此，我们建立了一种监测活细胞中 EGFP-Ub 标记的新合成蛋白质泛素化的方法，并研究了泛素 E1 酶抑制剂对新合成蛋白质的影响。我们的初步结果表明，FCCS 与点击反应的结合是研究活细胞中新合成蛋白质泛素化的有效策略。这种新方法可应用于蛋白质泛素化的基础研究和在活细胞水平进行药物筛选。
