CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2023 Aug 8;95(31):11751-11760. doi: 10.1021/acs.analchem.3c01903. Epub 2023 Jul 28.
The formation of amorphous misfolded and aggregated proteins is a hallmark of proteome stress in diseased cells. Given its lack of defined targeting sites, the rational design of intracellular proteome aggregation sensors has been challenging. Herein, we modulate the amphiphilicity of fluorescent protein chromophores to enable selective detection of aggregated proteins in different biological samples, including recombinant proteins, stressed live cells, intoxicated mouse liver tissue, and human hepatocellular carcinoma tissue. By tuning the number of hydroxyl groups, we optimize the selectivity of fluorescent protein chromophores toward aggregated proteins in these biological samples. In recombinant protein applications, the most hydrophobic P0 (cLogP = 5.28) offers the highest fold change (FC = 31.6), sensitivity (LLOD = 0.1 μM), and brightness (Φ = 0.20) upon binding to aggregated proteins. In contrast, P4 of balanced amphiphilicity (cLogP = 2.32) is required for selective detection of proteome stresses in live cells. In mouse and human liver histology tissues, hydrophobic P1 exhibits the best performance in staining the aggregated proteome. Overall, the amphiphilicity of fluorescent chromophores governs the sensor's performance by matching the diverse nature of different biological samples. Together with common extracellular amyloid sensors (e.g., Thioflavin T), these sensors developed herein for intracellular amorphous aggregation complement the toolbox to study protein aggregation.
无定形错误折叠和聚集的蛋白质的形成是病变细胞蛋白质组应激的标志。由于缺乏明确的靶向部位，因此合理设计细胞内蛋白质组聚集传感器一直具有挑战性。在这里，我们调节荧光蛋白生色团的两亲性，以实现对不同生物样品中聚集蛋白质的选择性检测，包括重组蛋白、应激活细胞、中毒的鼠肝组织和人肝癌组织。通过调整羟基的数量，我们优化了荧光蛋白生色团对这些生物样品中聚集蛋白质的选择性。在重组蛋白应用中，最疏水的 P0（cLogP = 5.28）在与聚集蛋白结合时提供最高的折叠变化（FC = 31.6）、灵敏度（LLOD = 0.1 μM）和亮度（Φ = 0.20）。相比之下，具有平衡两亲性的 P4（cLogP = 2.32）需要用于选择性检测活细胞中的蛋白质组应激。在鼠和人肝组织学组织中，疏水性 P1 在染色聚集蛋白质组方面表现出最佳性能。总的来说，荧光生色团的两亲性通过匹配不同生物样品的多样性来控制传感器的性能。与常见的细胞外淀粉样传感器（例如，硫黄素 T）一起，本文开发的用于细胞内无定形聚集的这些传感器补充了研究蛋白质聚集的工具包。
