All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.
Keywords: GAPDH; NAD biosynthesis; NAD compartmentalization; NAMPT; NMN/NAD+ salvage pathway; cell stress; melanoma; metabolism; nicotinamide adenine dinucleotide (NAD); nicotinamide mononucleotide (NMN); nucleus; protein-protein interaction; redox cycling.
© 2020 Grolla et al.