Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch
Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch
复制标题
DOI：
10.1021/acs.bioconjchem.9b00696
复制
发表时间：
2020-03-01
影响因子：
4.7
通讯作者：
Kikuchi, Kazuya
中科院分区：
文献类型：
--
作者：
Gao, Jingchi;Hori, Yuichiro;Kikuchi, Kazuya
蛋白质降解在细胞内稳态和信号转导中起着多种作用。实时监测降解过程不仅有助于阐明相关的生物学现象，而且为靶向蛋白质降解的药物发现提供了有力的工具。用蛋白质标签和合成荧光探针标记荧光蛋白是一种强大的技术，能够直接观察活细胞中的感兴趣蛋白质。虽然已经报道了各种蛋白质标签及其标记探针，但用于活细胞中蛋白质降解的可视化技术仍然有限。为了克服这种限制，我们在本文中采用了具有荧光关闭开关的PYP-标签标记探针，其能够对蛋白质降解进行成像。此外，我们进行了PYP标签的基于结构的设计，以稳定由探针和蛋白质标签形成的复合物，用于长期活细胞成像。我们成功地将该技术应用于Regnase-1响应免疫刺激的降解过程的活细胞成像。
Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP- tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.