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Biosensor reveals multiple sources for mitochondrial NAD+

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Science  17 Jun 2016:
Vol. 352, Issue 6292, pp. 1474-1477
DOI: 10.1126/science.aad5168

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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

Abstract

Nicotinamide adenine dinucleotide (NAD+) is an essential substrate for sirtuins and poly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD+-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD+ concentrations within these subcellular compartments are thought to regulate the activity of NAD+-consuming enzymes; however, the challenge in measuring compartmentalized NAD+ in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD+ concentrations in subcellular compartments. We found that the concentrations of free NAD+ in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD+ biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD+ concentrations.

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