Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch.,Bioconjugate Chemistry

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Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2019-12-26 , DOI: 10.1021/acs.bioconjchem.9b00696
Jingchi Gao , Yuichiro Hori , Osamu Takeuchi ₁ , Kazuya Kikuchi

Affiliation

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.

中文翻译：

利用设计的蛋白质标签突变体和带关闭开关的荧光探针对蛋白质降解进行活细胞成像。

蛋白质降解在细胞动态平衡和信号转导中起多种作用。降解过程的实时监控不仅有助于阐明相关的生物学现象，而且还为针对蛋白质降解的药物发现提供了强大的工具。带有蛋白质标签和合成荧光探针的荧光蛋白质标记是一项强大的技术，可直接在活细胞中可视化目标蛋白质。尽管已经报道了多种蛋白质标签及其标记探针，但是在活细胞中蛋白质降解可视化的技术仍然有限。为了克服此限制，我们在本文中采用了带有荧光关闭开关的PYP标签标记探针，该开关能够对蛋白质降解进行成像。此外，我们对PYP标签进行了基于结构的设计，以稳定由探针和蛋白质标签形成的复合物，以进行长期的活细胞成像。我们成功地将此技术应用于Regnase-1响应免疫刺激降解过程的活细胞成像。

更新日期：2020-01-09

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