Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.
Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.
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基因编码荧光传感器揭示 NADPH 代谢的动态调节
DOI：
10.1038/nmeth.4306
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发表时间：
2017-07
期刊：
影响因子：
48
通讯作者：
Yang Y
中科院分区：
文献类型：
--
作者：
Tao R;Zhao Y;Chu H;Wang A;Zhu J;Chen X;Zou Y;Shi M;Liu R;Su N;Du J;Zhou HM;Zhu L;Qian X;Liu H;Loscalzo J;Yang Y
还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）对于生物合成反应和抗氧化功能至关重要;然而，活细胞中NADPH代谢的检测仍然具有技术挑战性。我们开发并表征了具有各种亲和力和宽动态范围的NADPH（iNap传感器）的比率，pH抗性，遗传编码荧光指示剂。iNap传感器允许量化由胞质NAD+激酶水平控制的胞质和线粒体NADPH池，并揭示了氧化应激下取决于葡萄糖可用性的细胞NADPH动力学。我们发现，哺乳动物细胞有很强的倾向，以维持生理NADPH稳态，这是由葡萄糖-6-磷酸脱氢酶（G6 PD）和AMP激酶（AMPK）的调节。此外，使用iNap传感器，我们监测体内巨噬细胞激活或伤口反应期间的NADPH波动。这些数据表明，iNap传感器将成为监测活细胞中NADPH动力学的宝贵工具，并获得对细胞代谢的新见解。
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. The iNap sensors permitted quantification of cytosolic and mitochondrial NADPH pools that were controlled by cytosolic NAD+ kinase levels, and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We find that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) and AMP kinase (AMPK). Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live-cells, and gaining new insights into cell metabolism.
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影响因子：
16.6
作者：
通讯作者：
--
DOI：
10.1126/science.aad5168
发表时间：
2016-06-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Cambronne XA;Stewart ML;Kim D;Jones-Brunette AM;Morgan RK;Farrens DL;Cohen MS;Goodman RH
通讯作者：
Goodman RH
影响因子：
3
作者：
Bilan, Dmitry S.;Matlashov, Mikhail E.;Gorokhovatsky, Andrey Yu.;Schultz, Carsten;Enikolopov, Grigori;Belousov, Vsevolod V.
通讯作者：
Belousov, Vsevolod V.
影响因子：
29
作者：
Pavlova NN;Thompson CB
通讯作者：
Thompson CB
影响因子：
48
作者：
Cameron, William D.;Bui, Cindy V.;Rocheleau, Jonathan V.
通讯作者：
Rocheleau, Jonathan V.