Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metab

发布日期2017-09-26 08:56 来源:本站

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism

Tao R1Zhao Y1,2,3Chu H4Wang A1Zhu J1Chen X1,2,3Zou Y1Shi M1Liu R1Su N1Du J2Zhou HM5Zhu L6Qian X7Liu H4Loscalzo J8Yang Y1,2.

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.


https://www.nature.com/nmeth/journal/v14/n7/full/nmeth.4306.html