用于测量腺嘌呤核苷酸与AMPK结合的基于荧光共振能量转移的传感器。

FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK.

作者信息

Abi Nahed Roland, Pelosse Martin, Aulicino Francesco, Cottaz Florine, Berger Imre, Schlattner Uwe

机构信息

Univ. Grenoble Alpes, INSERM U1055, Laboratory of Fundamental and Applied Bioenergetics (LBFA), 2280 Rue de la Piscine, Domaine Universitaire Gières, Grenoble, France.

Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BSH 1TD, United Kingdom, Bristol, UK.

出版信息

Methods Mol Biol. 2025;2882:15-45. doi: 10.1007/978-1-0716-4284-9_2.

DOI:10.1007/978-1-0716-4284-9_2

PMID:39992503

Abstract

AMP-activated protein kinase (AMPK) has evolved to detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy supply. Such energy stress then leads to AMPK activation and downstream events that maintain cellular energy homeostasis. AMPK activation by AMP, ADP, or pharmacological activators involves a conformational switch within the AMPK heterotrimeric complex. We have engineered an AMPK-based sensor, AMPfret, which translates the activating conformational switch into a fluorescence signal, based on increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. Here we describe how this sensor can be used to analyze direct AMPK activation by small molecules in vitro using a fluorimeter, or to estimate changes in the energy state of cells using standard fluorescence or confocal microscopy.

摘要

AMP激活的蛋白激酶（AMPK）已经进化到能够检测细胞内AMP/ATP和ADP/ATP浓度比的关键增加，以此作为限制能量供应的信号。这种能量应激随后导致AMPK激活以及维持细胞能量稳态的下游事件。AMP、ADP或药理激活剂对AMPK的激活涉及AMPK异源三聚体复合物内的构象转换。我们设计了一种基于AMPK的传感器AMPfret，它基于供体和受体荧光团之间增强的荧光共振能量转移（FRET），将激活的构象转换转化为荧光信号。在这里，我们描述了如何使用该传感器通过荧光计在体外分析小分子对AMPK的直接激活，或使用标准荧光或共聚焦显微镜估计细胞能量状态的变化。