Abstract
We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD+) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP+ are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP+ ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.
Highlights
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are cofactors involved in a multitude of redox reactions regulating energy metabolism, reductive biosynthesis and antioxidant defense
We speculated that the -stacking interaction between p the sulfa drug and the nicotinamide moiety of NADP+ could be exploited to generate a semisynthetic biosensor for NADP+ (Figure 1a,b)
According to our design principle, the tethered sulfamethoxazole should bind to sepiapterin reductase (SPR) in an NADP+-dependent manner, thereby increasing FRET efficiency between the two fluorophores
Summary
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are cofactors involved in a multitude of redox reactions regulating energy metabolism, reductive biosynthesis and antioxidant defense. Free NAD(P)H/NAD(P)+ ratios can be indirectly determined by measuring the ratio of selected redox couples (Williamson et al, 1967; Veech et al, 1969). Such approaches lack spatial resolution and are not suitable for studying dynamic changes. Current sensors can measure changes in free NAD+/NADH ratio (Zhao et al, 2015), NADH (Zhao et al, 2011; Hung et al, 2011), NAD+ (Cambronne et al, 2016), NADP+ (Cameron et al, 2016) as well as NADPH (Tao et al, 2017)
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