the sirtuin deacetylases, the adenosine diphosphate (ADP)-ribose transferases/poly(ADP-ribose) polymerases, and the cyclic ADP-ribose (cADPR) synthases. The ensuing protein modification and messenger generation play a spectrum of roles in gene-regulatory, signaling, metabolic and cellular homeostasis pathways, and also in aging and diseases. There are three pathways of NAD utilization: the sirtuin mediated pathway and the ADP ribosylation pathways - mono and poly-ribosylation, and cyclic ribosylation. Competition between NAD-consuming enzymes for the limited shared substrate is envisioned. Particularly interesting is the competition between Sirt1 and Parp1, the better and best characterized members of their families. PARP1 activity increases with aging, as a result of increased DNA damage. The decrease in NAD concentration can reach 80% with maximal PARP1 activation. This can negatively impact on Sirtuin activity, whose function is thought to increase life span, among others. Evidence is emerging that SIRT1 and PARP1 are engaged in an antagonistic relationship whose balance benefits cells but whose alteration is detrimental. Details of the individual NAD utilization pathways are presented.

Sirtuin-mediated pathway
Sirtuins (Sirtuin 1-7), the NAD-dependent deacetylases, are members of class III deacetylases. Sirt1, 6 and 7 are nuclear proteins, Sirt2 is cytoplasmic but can translocate to the nucleus, Sirt3, 4 and 5 are mitochondrial proteins. In addition to deacetylation of histones and other proteins, they can carry out other reactions, such as decrotonylation, demalonylation, desuccinylation or deglutarylation. Sirt4, which does not seem to have deacetylase activity, can act as an ADP-ribosyltransferase, an activity also exhibited by Sirt6. All sirtuins have a conserved catalytic NAD binding domain of ~225 amino acids, flanked by variable length N- and C-termini that are targets for regulatory posttranslational modifications. Sirtuins are thought of as sensors of the NAD/nicotinamide (NAM) ratio. NAM, the product of the reaction, binds noncompetitively to sirtuins and inhibits them. The reduced form of NAD, NADH, has also been proposed to act as an inhibitor by competing with NAD for binding to sirtuins. Sirt1 is the largest and the best characterized of sirtuins; targets, in addition to histones, include many transcription factors along with transcriptional regulators and cofactors. Phosphorylation and methylation, sumoylation and nitrosylation of Sirt1 have been reported, also phosphorylation of other sirtuins. Sirt1 and 6 are involved in the circadian control of gene expression. Collectively sirtuins are involved in chromatin modification, transcription and gene expression regulation, mitochondria function and cellular homeostasis.

ADP ribosylation pathway, mono and poly-ribosylation
Mono and poly-ADP-ribosylation are carried out by members of poly(ADP)-ribose polymerases - PARPs which catalyze the transfer of the ADP-ribose moiety of NAD+ to protein acceptors. Usually these are glutamine and asparagine residues, but cysteine and lysine can also be modified. There are 17 members in the mammalian family and apart from the highly conserved ADP-ribosyl transferase (ART), catalytic domain show great structural domain variability. Most can only mono-ribosylate substrates (MAR), but a few can produce long chains (PAR), up to 200 units, and even introduce branching, occasionally. Some require DNA binding for full enzymatic activity, others, such as those with macrodomains, may not exhibit detectable enzymatic activity, eg PARP9, but can bind modified proteins. The macrodomain is one of the ADP-ribose biding modules; its presence in some PARP members renders them both writers (proteins that modify) and readers (proteins that recognize the modification). This domain is also present in the erasers (proteins that reverse the modification). Of all PARPs, the DNA-dependent PARP1 is the best characterized and also one of the more complex. Parp1 is among the few enzymes catalyzing poly-ribosylation (others include Parp2 and tankyrases), and can also induce branching; it recognizes double-strand breaks and other anomalous DNA structures, such as forks, hairpins, overhangs or cruciform structures. Readers of ADP-ribosylation contain ADP-ribose binding modules such as the macrodomain mentioned above, poly(ADP-ribose)-binding zinc finger (PBZ), or WWE domains. However, not too many readers have been identified and fully characterized. Erasers, such PAR glycohydrolase, the Macro1, Macro2 and Oard1 (TARG1) glycohydrolases, all have a macrodomain fold to bind the modified protein and catalyze ADP-ribosyl hydrolysis. Of these, only Parg can hydrolyze PARs. Responses elicited by ADP-ribose protein modification include DNA damage repair, transcription, cell cycle regulation and cell death.

ADP ribosylation pathway, cyclic ribosylation
Cd38 and Bst1/Cd157 are the enzymes involved in the formation of cyclic ADP-ribose (cADPR) and more is known about Cd38, a type II transmembrane glycoprotein although overall, less is known about this route of NAD consumption than the other, described above. cADPR prompts calcium release from intracellular stores. Another NAD derivative known to be a very potent calcium release agent is nicotinic acid adenine dinucleotide phosphate (NAADP), but the exact mode of its formation is rather debated. Among the several possible routes leading to NAADP is via conversion of 2-phospho-cADPR. Under acidic conditions, Cd38 may be able to form NAADP. Of note is the high NAD consumption by Cd38, with hydrolysis of ~100 NAD molecule to yield one cADPR molecule. As a type II transmembrane protein, Cd38 has an intracellular N-terminus and an extracellular C-terminus. The C-terminus contains the catalytic site and models for how NAD is processed extracellularly have included NAD transport and Cd38 expression as both type II and type III proteins, the latter having the opposite orientation. The C-terminus also houses 6 pairs of disulfide bonds; their formation within an intracellular milieu being generally doubted. However, a soluble cytosolic Cd38 construct has been shown to have catalytic activity and intact disulfide bonds. cADPR activates ryanodine receptors to prompt Ca release from the sarcoplasmic reticulum. NAADP prompts release of calcium from endolysosomal stores via two-pore channels.

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