Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Forster Resonance Energy Transfer Indicator
Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Forster Resonance Energy Transfer Indicator
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DOI：
10.1021/acs.analchem.0c01741
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发表时间：
2020-08-04
影响因子：
7.4
通讯作者：
San Martin, Alejandro
中科院分区：
文献类型：
--
作者：
Galaz, Alex;Cortes-Molina, Francisca;San Martin, Alejandro
乳酸和丙酮酸的胞浆浓度之比是糖酵解和线粒体氧化代谢之间平衡的直接读数。目前的方法不允许在生命系统中以高空间/时间分辨率在单一读数中检测乳酸/丙酮酸比率。使用基于Forster共振能量转移(FRET)的筛选策略，我们发现地衣芽孢杆菌的孤儿转录因子LutR是乳酸/丙酮酸比率的内源传感器，适合用作结合部分来开发基于FRET的遗传编码指示剂Lapronic。该指示剂对乳酸和丙酮酸的敏感性是通过改变荧光FRET比率来表征的，并用等温滴定量热法进行了验证。Lapronic对生理pH和温度不敏感，对结构相关的分子乙酸盐和β-羟基丁酸酯或辅因子NAD+和NADH不反应。Lapronic在HEK 293细胞中表达，表现出均一的胞浆定位，并且也针对线粒体基质。设计了一种校准方案来定量评估完整哺乳动物细胞中的乳酸/丙酮酸比率。从大肠杆菌中提纯的蛋白具有很强的稳定性，适合于生物样品中乳酸/丙酮酸比值的检测。我们预计，Lapronic将对基础和应用研究具有实际意义。
The ratio between the cytosolic concentrations of lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a Forster resonance energy transfer (FRET)-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable for use as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in the fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules acetate and beta-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK 293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed robust stability over time and was found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.