Portal de Periódicos da CAPES

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism

2017; Nature Portfolio; Volume: 14; Issue: 7 Linguagem: Inglês

10.1038/nmeth.4306

1548-7105

Rongkun Tao, Yuzheng Zhao, Huanyu Chu, Aoxue Wang, Jiahuan Zhu, Xianjun Chen, Yejun Zou, Mei Shi, Renmei Liu, Ni Su, Jiulin Du, Hai‐Meng Zhou, Linyong Zhu, Xuhong Qian, Haiyan Liu, Joseph Loscalzo, Yi Yang,

Immune cells in cancer

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential 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. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. 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.