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首页> 外文期刊>Cell metabolism >Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor.
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Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor.

机译：用遗传编码的荧光生物传感器成像胞质NADH-NAD（+）氧化还原状态。

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摘要

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] x [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.
机译：NADH是关键的代谢辅因子，很难在活细胞的细胞质中进行灵敏而特异的检测。我们通过结合环状排列的GFP T-蓝宝石与细菌NADH结合蛋白Rex，构建了细胞质NADH-NAD（+）氧化还原状态的荧光生物传感器。尽管最初的构建体报道了[NADH] x [H（+）] / [NAD（+）]，但通过诱变消除了其pH敏感性。工程化的生物传感器Peredox报告了细胞质中NADH：NAD（+）的比例，可以用外源性乳酸和丙酮酸进行校准。我们证明了其在几种培养和原代细胞类型中的效用。我们发现糖酵解反对乳酸脱氢酶平衡，以产生减少的胞质NADH-NAD（+）氧化还原状态。我们还观察到原代小鼠星形胶质细胞和神经元中不同的氧化还原状态，与假设的代谢差异一致。此外，使用高内涵图像分析，我们监测了数百种活细胞中NADH对PI3K途径抑制的反应。作为NADH的记者，Peredox应该能够更好地理解生物能学。

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