蛋白靶向的比率型谷胱甘肽荧光探针构建与亚细胞定量检测研究

Glutathione (GSH) is an important thiol compound, which is closely related to various physiological and pathological processes (e.g. redox regulation, cancer, inflammation, neurodegenerative disease, etc.). The real-time measurement of synthesis, distribution, metabolism and functional regulation of GSH at subcellular and protein level has great significance to deeply understand the GSH function. In this project, a protein-tag technology (SNAP-tag) is employed to achieve a specific labeling of target protein with organic small molecular fluorescent probes for ratiometric measurement of GSH. This method can be used for ratiometric imaging and quantitative detection of GSH in protein locations, which benefits from the high specificity and sensitivity of GSH probe. Meanwhile, the versatile SNAP-tag labeled with probes can be used for measurement of GSH in various organelles (mitochondria, nucleus, endoplasmic reticulum, peroxisomes, etc.) and proteins locations (glutaredoxin, thioredoxin, GSH reductase, etc.). Further, we will evaluate the relationship between GSH function and distribution in protein locations under different processes (ER stress, apoptosis, etc.). This project could provide a method for deeper exploration of subcellular GSH functional regulation.

谷胱甘肽（GSH）是细胞内重要的巯基化合物，它与众多的生理学和病理学过程（如氧化还原调控、癌症、炎症、神经退行性疾病等）密切相关。在亚细胞及蛋白质水平实时分析GSH的合成、分布、代谢和功能调控等复杂过程，对于其功能的深入研究具有重要意义。本项目拟通过蛋白标签技术（SNAP-tag），实现有机小分子比率型GSH荧光探针对靶标蛋白质的特异性标记。结合小分子探针特异性好、灵敏度高的优点，可实现蛋白质水平GSH的比率成像与定量检测。同时，凭借标记方法的通用性，可实现对多种GSH相关细胞器（线粒体、细胞核、内质网和过氧化物酶体等）和相关蛋白（谷氧还蛋白、硫氧还蛋白和谷胱甘肽还原酶等）的靶向标记及其GSH水平检测。研究将进一步在内质网应激和细胞凋亡等过程中评价GSH在蛋白质水平的分布与功能的关系。该项研究将为GSH亚细胞功能调控的深入研究提供手段。

谷胱甘肽（GSH）是细胞内重要的巯基化合物，它与众多的生理学和病理学过程（如氧化还原调控、癌症、炎症、神经退行性疾病等）密切相关。在亚细胞及蛋白质水平实时分析GSH的合成、分布、代谢和功能调控等复杂过程，对于其功能的深入研究具有重要意义。本项目首先构建了一系列特异性、选择性的GSH荧光探针，并将该类探针用于GSH的荧光检测与成像；随后构建了用于细胞内蛋白靶向标记的SNAP-tag融合标签体系，并利用一系列荧光染料的苄基鸟嘌呤衍生物实现了多种亚细胞（细胞质、细胞核、线粒体、内质网和过氧化物酶体等）蛋白质的特异性标记与荧光成像；最后构建了可用于蛋白标记的GSH荧光探针，实现了亚细胞特异性标记与GSH的比率成像。该项研究为GSH亚细胞功能调控的深入研究提供了手段。