Inhibition of Nicotinamide Phosphoribosyltransferase

Abstract

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly-and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.Several enzymes that transform NAD, used as a bona fide substrate, into metabolites displaying pleiotypic properties have been recently identified (1-4). Among them, ADP-ribose (ADPR) 2 transferases such as poly(ADP-ribose) polymerase (PARP)-1, -2, -3, -10, tankyrases, v-PARP, and others members of the same family are involved in processes such as nuclear homeostasis, cell differentiation, and death (5, 6). Mono(ADPribose) transferases (MART) are NAD-consuming enzymes present on cell membranes (but probably also in cytosol and organelles) that are still poorly understood (7). Additional NAD-consuming enzymes are CD-38/CD157, two ectoenzymes synthesizing ADPR, and the intracellular Ca 2ϩ -mobilizing compound cyclic ADPR (8), and sirtuins, a family of proteins encompassing seven members (Sirt1-7) involved in numerous processes such as chromatin homeostasis, transcription, cell death, and lifespan extension (9).Because of the constitutive activity of the NAD-consuming enzymes, eukaryotic cells evolved a rescue pathway leading to NAD resynthesis from nicotinamide (Nam). NAD rescue occurs in parallel to NAD neosynthesis from tryptophan or nicotinic acid (the so-called "kynurenine" and "Priess-Handler" pathways, respectively) (10, 11). Recently, work from the Brenner laboratory (12) identified nicotinamide riboside as an additional NAD precursor. The biochemical route of NAD rescue is composed by two enzymatic steps, the first operated by nicotinamide phosphoribosyltransferase (Nampt), forming nicotinamide mononucleotide from Nam and phosphoribosyl pyrophosphate, and the second driv...

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

“…1A). However, the presence of NamPRT in mitochondria was not confirmed in other studies (28,29). Consequently, a conclusive assessment of the presence of NamPRT and NMNAT3 in mitochondria is required to understand NAD ϩ generation within these organelles.…”

“…Mitochondrial NAD ϩ -dependent protein deacetylation and mono-ADP-ribosylation regulate key metabolic enzymes, including glutamate dehydrogenase (25,26), acetyl-CoA synthetase 2 (27), and carbamoyl-phosphate synthetase 1 (28). The mitochondrial NAD ϩ pool is autonomous (29) and appears to be most critical for cell survival (30). In yeast and plants, cytosolic NAD ϩ is imported across the inner mitochondrial membrane (31,32), whereas mammalian mitochondria need to take up a precursor from the cytosol, which can be converted into NAD ϩ within the organelles.…”

NAD+ Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus

“…In addition, the presence of different forms of NMNATs in each cellular compartment (e.g., NMNAT1 in the nucleus or NMNAT3 in the mitochondria/cytosol) suggests that NAD + salvage is tailored according to compartment-specific metabolic needs. However, despite some evidence that NAMPT is localized to the mitochondria (Yang et al, 2007a), there is still some debate as to whether this is really the case (Pittelli et al, 2010). Therefore further experimental evidence is needed to confirm mitochondrial NAD + salvage.…”

“…As discussed in section 2.3, cytoplasmic NAD + levels cannot alter mitochondrial NAD + /NADH ratios directly since NAD + is not permeable to the mitochondrial membrane (Barile et al, 1996). Therefore, changes in the cytoplasmic NAD + pool do not acutely alter mitochondrial NAD + levels (Pittelli et al, 2010; Yang et al, 2007a). …”

Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio

“…Mitochondria maintain NAD + levels during genotoxic stress and promote cell survival even when NAD + in the nucleus and cytosol have fallen well below normal physiological levels [23]. Additionally, chronic treatment of FK866 – a potent inhibitor of NAMPT – was found to deplete cytosolic but not mitochondrial NAD + [24]. Thus, partitioning discrete dinucleotide pools allows cells to maintain NAD + levels under environmental, pharmacological, or genetic stressors.…”