Nicotinamide mononucleotide adenylyltransferase (NMNAT) is the central enzyme of the NAD biosynthetic pathway. Three human NMNAT isoforms have recently been identified, but isoform-specific functions are presently unknown, although a tissue-specific role has been suggested. Analyses of the subcellular localization confirmed NMNAT1 to be a nuclear protein, whereas NMNAT2 and -3 were localized to the Golgi complex and the mitochondria, respectively. This differential subcellular localization points to an organelle-specific, nonredundant function of each of the three proteins. Comparison of the kinetic properties showed that particularly NMNAT3 exhibits a high tolerance toward substrate modifications. Moreover, as opposed to preferred NAD ؉ synthesis by NMNAT1, the other two isoforms could also form NADH directly from the reduced nicotinamide mononucleotide, supporting a hitherto unknown pathway of NAD generation. A variety of physiological intermediates was tested and exerted only minor influence on the catalytic activities of the NMNATs. However, gallotannin was found to be a potent inhibitor, thereby compromising its use as a specific inhibitor of poly-ADP-ribose glycohydrolase. The presence of substrate-specific and independent nuclear, mitochondrial, and Golgi-specific NAD biosynthetic pathways is opposed to the assumption of a general cellular NAD pool. Their existence appears to be consistent with important compartment-specific functions rather than to reflect simple functional redundance.For many years the pyridine nucleotides have been recognized as vital to all organisms because of their essential role in energy transduction. They participate as coenzymes in a multitude of metabolic hydrogen transfer reactions. Recently, however, an entirely unexpected role of NAD and NADP has been discovered that is now the subject of intense investigations; these molecules represent important factors in a variety of signaling pathways (1). On the one hand, NAD ϩ is a substrate for covalent protein modifications such as mono-ADP-ribosylation, poly-ADP-ribosylation, and protein deacetylation. These processes participate in the regulation of critical cellular events such as replication, telomere maintenance, apoptosis, and transcription as well as gene silencing (1-5). On the other hand, NAD(P) ϩ is the direct precursor of two calcium-mobilizing messengers, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). 3 These molecules are involved in a variety of signaling pathways including the regulation of intracellular insulin levels and its secretion, T-cell activation, or catecholamine secretion (6 -9). The discovery of NAD-dependent regulatory mechanisms has also led to the realization that NAD(P) biosynthesis has to be an ongoing process to fuel these pathways. That is, although the metabolic redox reactions are not accompanied by any net consumption of pyridine nucleotides, their participation in signaling pathways requires a steady resynthesis to maintain a stable cellular concentration.Nicotinami...

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Nuclear NAD ؉ metabolism constitutes a major component of signaling pathways. It includes NAD ؉ -dependent protein deacetylation by members of the Sir2 family and protein modification by poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 has emerged as an important mediator of processes involving DNA rearrangements. High-affinity binding to breaks in DNA activates PARP-1, which attaches poly(ADP-ribose) (PAR) to target proteins. NMN adenylyl transferases (NMNATs) catalyze the final step of NAD ؉ biosynthesis. We report here that the nuclear isoform NMNAT-1 stimulates PARP-1 activity and binds to PAR. Its overexpression in HeLa cells promotes the relocation of apoptosis-inducing factor from the mitochondria to the nucleus, a process known to depend on poly(ADP-ribosyl)ation. Moreover, NMNAT-1 is subject to phosphorylation by protein kinase C, resulting in reduced binding to PAR. Mimicking phosphorylation, substitution of the target serine residue by aspartate precludes PAR binding and stimulation of PARP-1. We conclude that, depending on its state of phosphorylation, NMNAT-1 binds to activated, automodifying PARP-1 and thereby amplifies poly(ADP-ribosyl)ation.apoptosis ͉ NAD biosynthesis ͉ poly(ADP-ribosyl)ation ͉ protein phosphorylation N MN adenylyl transferase (NMNAT) is an essential enzyme because it catalyzes the final step of NAD ϩ biosynthesis (1). There are three isoforms in humans that exhibit tissue-and organelle-specific expression (2). Because the biological role of NAD ϩ and NADH (collectively termed NAD) has long been thought to be confined to their function as electron carriers, the nuclear localization of the major isoforms, human NMNAT-1 (3) and yeast Nma2 (4), was somewhat puzzling. However, recent research has revealed many important regulatory functions of the pyridine nucleotides, some of which take place within the nucleus (5).NAD ϩ serves as substrate for covalent protein modifications such as mono(ADP-ribosyl)ation (6), poly(ADP-ribosyl)ation (7,8), and protein deacetylation by sirtuins, proteins of the silent information regulator 2 (Sir2) family (9). Pyridine nucleotides are also direct precursors of two calcium-mobilizing messengers, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP ϩ ) (10 -13). The predominant NAD ϩ -consuming activity, poly(ADP-ribosyl)ation by poly(ADPribose) polymerase 1 (PARP-1), occurs within the nucleus, as does NAD ϩ -dependent protein deacetylation by sirtuins. Consequently, substrate supply by NMNAT-1 may directly influence these processes. Overexpression of NMNAT-1 extends the lifespan of eukaryotic cells, which has been attributed to augmented NAD ϩ -dependent histone deacetylation catalyzed by Sir2p or its homologs (4,(14)(15)(16)(17)(18). Increased NMNAT activity is also required for the axon-sparing activity of the chimeric slow Wallerian degeneration (Wld s ) protein, which is composed of 19 amino acids of the ubiquitin assembly protein Ufd2a and full-length NMNAT-1 (19).Although suggested previously (3, 20, 21), a functional intera...

Calcium signaling is essential for the differentiation of many cell types, including skeletal muscle cells, but its mechanisms remain elusive. Here we demonstrate a crucial role for nicotinic acid adenine dinucleotide phosphate (NAADP) signaling in skeletal muscle differentiation. Although the inositol trisphosphate pathway may have a partial role to play in this process, the ryanodine signaling cascade is not involved. In both skeletal muscle precursors and C2C12, cells interfering with NAADP signaling prevented differentiation, whereas promoting NAADP signaling potentiated differentiation. Moreover, siRNA knockdown of two-pore channels, the target of NAADP, attenuated differentiation. The data presented here strongly suggest that in myoblasts, NAADP acts at acidic organelles on the recently discovered two-pore channels to promote differentiation.