Nicotinamide phosphoribosyltransferase (NAMPT) translocates to the nucleus following glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction and confers DNA damage resistance to melanoma cells.

Background:Some proteins, referred to as “moonlighting”, involved in cellular basic metabolism possess functions beyond the enzymatic ones required in energy generation. When these moonlighting proteins are up-regulated for metabolic reprogramming in cancer, a side-effect is given by the up-regulation of further cellular functions, which may also participate in cancer progression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is classified as such protein, which acquires new functions due to a change in oligomerization and/or subcellular localization. Indeed, it is involved in DNA repair, tRNA export, membrane fusion and transport, cytoskeletal dynamics. I have serendipitously discovered that GAPDH interacts with nicotinamide phosphoribosyl transferase (NAMPT). NAMPT catalyses the production of nicotinamide mononucleotide (NMN) and is the rate-limiting reaction in NAD biosynthesis. I demonstrated that GAPDH and NAMPT form a direct complex mainly in cancer cells and the complex is necessary for their translocation to the nucleus. Hypothesis:The hypothesis is that in cancer, the consequence of the up-regulation of moonlighting proteins involved in metabolism yields collateral effects given by their secondary actions, which are amenable to pharmacological intervention. Methods:We demonstrated NAMPT and GAPDH interaction both in cells via immunoprecipitation, and in recombinant proteins via pull-down assays and Surface Plasmon Resonance. The Small Angle X-ray Scattering and XL-MS analysis demonstrated how a dimer of NAMPT interacts with a monomer of GAPDH. The oxidative stress and DNA damage were induced via ROS and NO donors, UV radiations and alkylating agents. Results:We found a strong nuclear staining of NAMPT in melanoma lesions and a determinant of the outcome of the complex formation is given by nuclear localization. Interestingly, under a stress stimulus, the movement of both proteins through the nucleus resulted increased. Both the silencing of GAPDH and the treatment with omigapil, a specific inhibitor of nuclear GAPDH transport, induced a significant reduction of NAMPT into the nucleus. When we mutated NAMPT to not bind to GAPDH, the entire complex did not translocate to the nucleus. In consequence of NAMPT/GAPDH reduction in the nucleus, also NMN and NAD amount were reduced at that level. Surprisingly, we found the complex presence on chromatin, where resulted increased after DNA damage. Moreover, melanoma cells expressing the mutated form of NAMPT resulted more sensitive to etoposide compared to WT cells expressing, both in vitro and in vivo. Conclusions:NAMPT and GAPDH interact in cancer cells and the complex translocates to the nucleus where acquires a secondary function to sustain cell viability after DNA damage. The prevention of the complex formation with specific interfering peptides may be a pharmacological strategy specific for cancer cells, as it does not target the primary enzymatic activity of these ubiquitous proteins.