Nicotinamide adenine dinucleotide (NAD+) is best known as a cofactor in metabolic processes, but lately it has come into the limelight as a substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), enzymes that regulate protein deacetylation and DNA repair. The subcellular colocalization of NAD+-generating and NAD+-consuming enzymes suggests that the activity of NAD+-dependent enzymes may be regulated by localized fluctuation in NAD+, but this was not experimentally proven. To measure subcellular pools of free NAD+, a team from the Vollum Institute in Oregon led by Richard Goodman has now generated a cellular-compartment-specific NAD+ biosensor.

Cellular levels of NAD+ are maintained by the cooperative action of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenyl-transferases (NMNATs), which recycle components of NAD+ breakdown to regenerate the active form of the coenzyme. The final step of NAD+ biogenesis is catalyzed by three enzyme isoforms, each specific to a cell compartment—nucleus, cytoplasm or mitochondria.

To study NAD+ levels, Goodman's team fused Venus fluorescent protein to two NAD+ binding sites from bacterial DNA ligase (Cambronne et al., 2016). The resulting chimeric protein can bind NAD+ but cannot consume it. When it is attached to NAD+, its fluorescence is quenched at only one of two excitation peaks, allowing measurements of free NAD+ to be normalized to sensor expression level. Precise quantification of NAD+ levels using sensor targeted to different compartments of mammalian cells showed that mitochondria contain more than twice as much free NAD+ as other compartments. The authors also show that gene silencing and pharmacologic inhibition of NAMPT led to a decrease in NAD+ concentration in all compartments studied, but depletion of mitochondrial NAD+ occurred at a slower rate. By systematically silencing the expression of each NMNAT isoform, the team demonstrated that the cytoplasmic and nuclear pools are readily exchangeable, whereas at least two mechanisms maintain mitochondrial NAD+: import from the cytoplasm and NAD+ biogenesis by mitochondrial isoform NMNAT3.

Some NAD+ can be phosphorylated into NADP+, which has different roles in metabolism. A team from the University of Toronto recently generated a NADP+ biosensor that can offer new insights into a variety of diseases and conditions, highlighting the importance of small-molecule probes in metabolic studies (Cameron et al., 2016). Similarly, as NAD+-consuming enzymes are implicated in immunity, metabolism and lifespan control, the NAD+ sensor holds promise to advance the study of disease and aging.