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一种用于在细胞系统中实时可视化短寿命蛋白质降解的“关-开-关”荧光蛋白标记探针。

An "OFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems.

作者信息

Reja Shahi Imam, Hori Yuichiro, Kamikawa Takuya, Yamasaki Kohei, Nishiura Miyako, Bull Steven D, Kikuchi Kazuya

机构信息

Division of Applied Chemistry, Graduate School of Engineering, Osaka University Suita Osaka 565-0871 Japan

Immunology Frontier Research Center, Osaka University Suita Osaka 565-0871 Japan.

出版信息

Chem Sci. 2022 Jan 11;13(5):1419-1427. doi: 10.1039/d1sc06274c. eCollection 2022 Feb 2.

DOI:10.1039/d1sc06274c
PMID:35222926
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8809410/
Abstract

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

摘要

监测从细胞中清除不需要的和受损蛋白质的蛋白水解途径的能力，对于理解用于维持细胞稳态的多种过程至关重要。在本研究中，我们开发了一种新的蛋白质标记探针，该探针采用“关-开-关”荧光开关，以实现对活细胞中PYP标签蛋白构建体的表达（荧光开启）和降解（荧光关闭）进行实时成像。荧光开关由未结合探针中的分子内接触猝灭相互作用调节（荧光关闭），该相互作用在与PYP标签蛋白结合时被破坏，从而使荧光开启。当PYP标签-探针复合物发生蛋白水解降解时，猝灭作用恢复，导致荧光关闭。对探针结构和PYP标签突变体的优化，使得这种快速反应的“关-开-关”探针能够用于对短命蛋白质的表达和降解进行荧光成像。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/ef93de9898a4/d1sc06274c-f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/fd7ad8cbb541/d1sc06274c-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/2f457d95629b/d1sc06274c-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/af3cea4c687f/d1sc06274c-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/5a9f9b1d8126/d1sc06274c-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/7c07360b1df2/d1sc06274c-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/d0f59bb05694/d1sc06274c-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/64f714f5ecc3/d1sc06274c-f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/ef93de9898a4/d1sc06274c-f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/fd7ad8cbb541/d1sc06274c-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/2f457d95629b/d1sc06274c-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/af3cea4c687f/d1sc06274c-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/5a9f9b1d8126/d1sc06274c-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/7c07360b1df2/d1sc06274c-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/d0f59bb05694/d1sc06274c-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/64f714f5ecc3/d1sc06274c-f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/544c/8809410/ef93de9898a4/d1sc06274c-f8.jpg

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