利用黏度敏感荧光寿命探针探索内质网功能障碍对蛋白质相分离的影响

IF 6.7 1区 化学 Q1 CHEMISTRY, ANALYTICAL
Yu Wei, Xiangyu Zi, Jia Zhai, Man Zhang, Jiaqi Li, Zhenglong Sun, Minzi Ju*, Xin Zhang* and Baoxing Shen*, 
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引用次数: 0

摘要

退行性疾病与蛋白质相变密切相关。内质网(Endoplasmic reticulum, ER)是蛋白质合成的主要部位,其稳态失衡是蛋白质相变的关键触发因素。监测内质网微环境变化的有效工具对于研究蛋白质相行为至关重要。在这项工作中,我们开发了一套基于二氰亚甲基- 4h -吡喃的粘度敏感探针(即VisDCM探针),其荧光强度和荧光寿命对局部粘度变化具有双重响应。计算分析表明,在特定粘度下,VisDCM探针的荧光激活是由于受限的可达锥交机制。设计了针对内质网和目标蛋白的双色探针。他们揭示了内质网应激如何通过Ca2+信号调节TDP-43蛋白相分离。体外实验表明,TDP-43相分离是Ca2+依赖性的。Ca2+的增加促进了TDP-43液-液相分离和聚集。最后,利用荧光寿命成像技术绘制ers诱导的微环境变化。总之,这项工作提供了一个新的工具箱来可视化蛋白质相变,以及突出Ca2+在TDP-43相分离和聚集中的作用,为退行性疾病提供见解和潜在的治疗方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Exploring Endoplasmic Reticulum Dysfunction on Protein Phase Separation Using Viscosity-Sensitive Fluorescent Lifetime Probe

Exploring Endoplasmic Reticulum Dysfunction on Protein Phase Separation Using Viscosity-Sensitive Fluorescent Lifetime Probe

Degenerative diseases are closely associated with protein phase transitions. Endoplasmic reticulum (ER), the primary site of protein synthesis, experiences homeostasis imbalance as the key trigger of the protein phase transition. Effective tools to monitor ER microenvironment changes are crucial for investigating protein phase behavior. In this work, we developed a set of viscosity-sensitive probes based on dicyanomethylene-4H-pyran (namely, VisDCM probes) with dual response of fluorescence intensity and fluorescence lifetime to local viscosity changes. Computational analysis demonstrated that fluorescence activation of VisDCM probes is due to the restricted accessible conical intersection mechanism under specific viscosity. Dual-color probes targeting the ER and protein of interest were designed. They revealed how ER stress regulates TDP-43 protein phase separation via Ca2+ signaling. In vitro experiments exhibited that TDP-43 phase separation is Ca2+-dependent. Increased Ca2+ promotes TDP-43 liquid–liquid phase separation and aggregation. Finally, fluorescence lifetime imaging was applied to map ERS-induced microenvironment changes. In summary, this work provides a novel toolbox to visualize protein phase transitions as well as highlights Ca2+ role in TDP-43 phase separation and aggregation, offering insights and potential therapies for degenerative diseases.

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来源期刊
Analytical Chemistry
Analytical Chemistry 化学-分析化学
CiteScore
12.10
自引率
12.20%
发文量
1949
审稿时长
1.4 months
期刊介绍: Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.
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