Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

Abstract

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly-(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADPribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.NAD is an essential electron carrier and a key molecule of signaling pathways (1-4). In bioenergetic pathways, NAD is reversibly converted between its oxidized (NAD ϩ ) and reduced (NADH) states, which would not require continuous regeneration. Indeed, when the principal pathway of NAD ϩ synthesis from nicotinic acid (NA) 2 had been established (5), the "case" was nearly closed, because neither additional roles of NAD nor a regulatory importance of its synthesis were suspected. Meanwhile, discoveries of signaling processes in which NAD ϩ is degraded have dramatically changed this view. Signaling conversions of NAD ϩ include the cleavage to nicotinamide (Nam), which is recycled into NAD ϩ synthesis, and a concomitant reaction of the remaining ADP-ribose moiety. NAD ϩ -dependent deacetylases (members of the sirtuin family) and mono-ADP-ribosyltransferases control life span, the biological clock, insulin secretion, and key metabolic enzymes (6 -9). In addition, NAD ϩ represents the substrate for poly(ADP-ribosylation) to regulate DNA repair, transcription, telomerase activity, and chromatin dynamics (10 -12). NAD ϩ is also the precursor of cyclic ADP-ribose and NAADP, potent agents to mobilize calcium from intracellular stores (13). This multitude of NAD ϩ -degrading reactions clearly necessitates permanent re...

Nicotinamide Phosphoribosyltransferase in Malignancy: A Review

“…However, other investigators have not found mitochondrial Nampt. For example, Nikiforov et al 16 found no mitochondrial iNampt and instead found that the mitochondrial-specific Nmnat3 produced all the mitochondrial NAD from NMN imported from the cytosol. The reason for this discrepancy is unknown, but it may be due to using different cells, or different mitochondrial isolation and analysis techniques.…”

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

“…We also found that most endogenous Nmnat3 proteins reside in the cytoplasm (Hikosaka et al., 2014; Yamamoto et al., 2016), although several reports claim that Nmnat3 is present in the mitochondria (Nikiforov et al., 2011; VanLinden et al., 2015). However, we also reported that overexpression of Nmnat3 in HeLa cells encouraged localization of Nmnat3 in mitochondria (Hikosaka et al., 2014).…”

“…Nampt catalyzes the formation of NMN from nicotinamide (NAM) and 5‐phosphoribosyl‐pyrophosphate (PRPP), and NMN is subsequently converted to NAD by nicotinamide mononucleotide adenylyltransferase (Nmnat; Berger, Lau, Dahlmann, & Ziegler, 2005; Nikiforov, Dolle, Niere, & Ziegler, 2011). However, it remains unclear which NAD synthesis enzyme can be exploited to boost NAD levels in vivo.…”

“…In mammals, there are three Nmnat isozymes encoded by different genes, namely, Nmnat1, Nmnat2, and Nmnat3 (Berger et al., 2005). In particular, Nmnat3 has been found in the mitochondria and is widely associated with mitochondrial NAD biosynthesis (Berger et al., 2005; Nikiforov et al., 2011; VanLinden et al., 2015). Although Nmnat3 deficiencies in mice caused no obvious changes in mitochondrial NAD levels, suggesting redundancy between mammalian Nmnat isozymes (Hikosaka et al., 2014; Yamamoto et al., 2016), overexpression of Nmnat3 in human cultured cells reportedly contributed to mitochondrial NAD synthesis (Nikiforov et al., 2011).…”

Alteration of Neuron-Glia Interactions in Neurodegeneration: Molecular Biomarkers and Therapeutic Strategy

“…Extracelullar nucleotides (e.g. NAD + and NMN) undergo extracellular degradation resulting in the formation of permeable precursors which are further converted to NAD + in mitochondria due to activity of NMNAT3 localized to the mitochondrial matrix (Nikiforov et al, 2011). Interestingly enough, in genomewide screen for late-onset Alzheimer's disease, SNP of the NMNAT-3 gene was found, thus suggesting involvement of NAD + synthesizing pathways in pathogenesis of this neurodegenerative disorder ].…”