A fluorescent sensor for NAD ⁺ in living cells: Roles of cellular nicotinamide adenine dinucleotide (NAD ⁺ ) in metabolism, aging, and disease have garnered much interest, but methods have been lacking to measure the amounts of NAD ⁺ in living cells. Cambronne et al. developed a genetically encoded biosensor that can be used to monitor concentrations of free NAD ⁺ in various compartments of a cell (see the Perspective by Guarente). Such concentrations of NAD ⁺ appear to be important in regulating the activity of NAD ⁺ -consuming enzymes such as sirtuins and ADP-ribosyltransferases. The authors used the sensor to demonstrate that NAD ⁺ concentrations in mitochondria of cultured human cells can be controlled by multiple mechanisms. Science , this issue p. 1474 ; see also p. 1396

Nicotinamide adenine dinucleotide (NAD⁺) is an essential cofactor for redox enzymes, but also moonlights as a substrate for signaling enzymes. When used as a substrate by signaling enzymes, it is consumed, necessitating the recycling of NAD⁺ consumption products (i.e., nicotinamide) via a salvage pathway in order to maintain NAD⁺ homeostasis. A major family of NAD⁺ consumers in mammalian cells are poly-ADP-ribose-polymerases (PARPs). PARPs comprise a family of 17 enzymes in humans, 16 of which catalyze the transfer of ADP-ribose from NAD⁺ to macromolecular targets (namely, proteins, but also DNA and RNA). Because PARPs and the NAD⁺ biosynthetic enzymes are subcellularly localized, an emerging concept is that the activity of PARPs and other NAD⁺ consumers are regulated in a compartmentalized manner. In this review, I discuss NAD⁺ metabolism, how different subcellular pools of NAD⁺ are established and regulated, and how free NAD⁺ levels can control signaling by PARPs and redox metabolism.

ADP-ribosyltransferases (ARTD1-16) have emerged as major downstream effectors of NAD(+) signaling in the cell. Most ARTDs (ARTD7 and 8, 10-12, and 14-17) catalyze the transfer of a single unit of ADP-ribose from NAD(+) to target proteins, a process known as mono-ADP-ribosylation (MARylation). Progress in understanding the cellular functions of MARylation has been limited by the inability to identify the direct targets for individual mono-ARTDs. Here, we engineered mono-ARTDs to use an NAD(+) analog that is orthogonal to wild-type ARTDs. We profiled the MARylomes of ARTD10 and ARTD11 in vitro, identifying isoform-specific targets and revealing a potential role for ARTD11 in nuclear pore complex biology. We found that ARTD11 targeting is dependent on both its regulatory and catalytic domains, which has important implications for how ARTDs recognize their targets. We anticipate that our chemical genetic strategy will be generalizable to all mono-ARTD family members based on the similarity of the mono-ARTD catalytic domains.