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用HiBiT和NanoBRET对蛋白质循环和无处不在的定量见解

Małgorzata Piechota¹, Wojciech Pokrzywa¹

¹Laboratory of Protein Metabolism, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.

Methods in enzymology

|September 24, 2025

概括

我们优化了HiBiT和NanoBRET试验,以研究蛋白质稳定性和无处不在性. 这种方法提高了对林-RING E3酶功能和终端降解因子在向蛋白质降解中的作用的理解.

科学领域:

细胞生物学细胞生物学
生物化学生物化学
分子生物学分子生物学

背景情况:

蛋白质的循环和无处不在对于细胞平衡和蛋白质平衡至关重要.
了解向蛋白质降解途径对于细胞调节至关重要.

研究的目的:

通过使用HiBiT和NanoBRET测试来研究蛋白质稳定性,无处不在的动态和降解功能的优化方法.
研究Cullin-RING E3结合酶受体,包括VHL和FBXL15.
分析终端降解物对蛋白质稳定性和周转的影响.

主要方法:

利用HiBiT (11-氨基酸标签) 来实时量化蛋白质丰度和降解动力学.
采用NanoBRET (生物发光共振能量转移BRET) 来对活细胞监测无处不在的事件.
应用HiBiT标记来掩盖终端降解子,用于对N-和C-终端降解子功能的控制分析.

主要成果:

使用FBXL15作为模型证明,降解蛋白质可访问性显著影响蛋白质周转率.
在使用VHL作为案例研究的活细胞兼容系统中评估了无处不在的动态.
确立了HiBiT和NanoBRET作为调查库林-RING受体稳定性和终端降解功能的多功能工具.

结论:

HiBiT和NanoBRET提供了一种定量和生理学相关的方法资源,用于剖析蛋白质周转和无处不在的途径.
该研究提供了对翻译后修改和向蛋白质降解机制的见解.
优化的试验方便了对E3酶相互作用和基质无化效率的研究.

关键词:

库林-RING结合酶活细胞降解试验 Degrons 是一个衰退.在HiBiT稳定性分析中,在NanoBRET中使用NanoBRET.蛋白质的营业额无处不在化动态的动态

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