A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD + salvage pathway in the nucleus

J Biol Chem. 2020 Mar 13;295(11):3635-3651. doi: 10.1074/jbc.RA119.010571. Epub 2020 Jan 27.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Nucleus / enzymology*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / metabolism*
  • HeLa Cells
  • Humans
  • Kinetics
  • Melanoma, Experimental / enzymology
  • Melanoma, Experimental / pathology
  • Mice
  • NAD / metabolism*
  • NIH 3T3 Cells
  • Nicotinamide Mononucleotide / chemistry
  • Nicotinamide Mononucleotide / metabolism*
  • Nicotinamide Phosphoribosyltransferase / chemistry
  • Nicotinamide Phosphoribosyltransferase / metabolism*
  • Protein Binding
  • Protein Multimerization
  • Protein Transport
  • Stress, Physiological*

Substances

  • NAD
  • Nicotinamide Mononucleotide
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
  • Nicotinamide Phosphoribosyltransferase

Associated data

  • PDB/5U2M
  • PDB/5C7I