NAD + is well known as a crucial cofactor in the redox balance of metabolism. Moreover, NAD + is degraded in ADP-ribosyl transfer reactions, which are important components of multitudinous signalling reactions. These include reactions linked to DNA repair and aging. In the present study, using the concept of EFMs (elementary flux modes), we established all of the potential routes in a network describing NAD + biosynthesis and degradation. All known biosynthetic pathways, which include de novo synthesis starting from tryptophan as well as the classical Preiss–Handler pathway and NAD + synthesis from other vitamin precursors, were detected as EFMs. Moreover, several EFMs were found that degrade NAD + , represent futile cycles or have other functionalities. The systematic analysis and comparison of the networks specific for yeast and humans document significant differences between species with regard to the use of precursors, biosynthetic routes and NAD + -dependent signalling.
The pyridine nucleotides NAD and NADP play vital roles in metabolic conversions as signal transducers and in cellular defence systems. Both coenzymes participate as electron carriers in energy transduction and biosynthetic processes. Their oxidized forms, NAD + and NADP + , have been identified as important elements of regulatory pathways. In particular, NAD + serves as a substrate for ADP-ribosylation reactions and for the Sir2 family of NAD + -dependent protein deacetylases as well as a precursor of the calcium mobilizing molecule cADPr (cyclic ADP-ribose). The conversions of NADP + into the 2′-phosphorylated form of cADPr or to its nicotinic acid derivative, NAADP, also result in the formation of potent intracellular calcium-signalling agents. Perhaps, the most critical function of NADP is in the maintenance of a pool of reducing equivalents which is essential to counteract oxidative damage and for other detoxifying reactions. It is well known that the NADPH/NADP + ratio is usually kept high, in favour of the reduced form. Research within the past few years has revealed important insights into how the NADPH pool is generated and maintained in different subcellular compartments. Moreover, tremendous progress in the molecular characterization of NAD kinases has established these enzymes as vital factors for cell survival. In the present review, we summarize recent advances in the understanding of the biosynthesis and signalling functions of NAD(P) and highlight the new insights into the molecular mechanisms of NADPH generation and their roles in cell physiology.
NAADP (nicotinic acid–adenine dinucleotide phosphate) is a newly described intracellular messenger molecule that mediates Ca 2+ increases in a variety of cells. However, little is known of the mechanism whereby ligand binding regulates the target protein. We report in the present paper that NAADP receptors from sea urchin eggs undergo an unusual stabilization process that appears to be dependent upon the time during which receptors are exposed to their ligand. We demonstrate that receptors ‘tagged’ with NAADP for short periods were more readily dissociated following subsequent delipidation than those labelled for longer. Stabilization of NAADP receptors by their ligand was delayed relative to ligand association taking on the order of minutes to develop at picomolar concentrations. The stabilizing effects of NAADP did not require cytosolic factors or the continued presence of NAADP and persisted upon solubilization. NAADP receptors, however, failed to stabilize at reduced temperature. We conclude that NAADP receptors possess a simple molecular memory endowing them with the remarkable ability to detect the duration of their activation.
NAD + and its metabolites serve important functions in intracellular signalling. NAD + -mediated regulatory processes also take place on the cell surface, particularly of immune cells. In this issue of the Biochemical Journal , Gerth et al. have demonstrated a new mechanism of Ca 2+ uptake into monocytes which is triggered by NAD + or its degradation product, ADP-ribose. These observations point to a hitherto unknown Ca 2+ -influx mechanism and underscore the potential significance of NAD + and ADP-ribose as signalling molecules on the extracellular side of the plasma membrane.
NAD + glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca 2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [ 32 P]NAD + as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [ 32 P]NAD + only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 µ M) ADP-ribosylation was efficient with NAD + , but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD + by NAD + glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD + as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N -ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.
The present investigation identifies bovine liver mitochondrial NADase (NAD + glycohydrolase) as a member of the class of bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyse the synthesis and degradation of cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent. The mitochondrial enzyme utilized the NAD + analogues nicotinamide guanine dinucleotide (NGD + ) and nicotinamide hypoxanthine dinucleotide (NHD + ) to form fluorescent cyclic purine nucleoside diphosphoriboses. ADP-ribosyl cyclase activity was also demonstrated using 32 P-labelled NAD + as substrate. The identity of NADase and ADP-ribosyl cyclase was supported by their co-migration in SDS/polyacrylamide gels. Cyclase activity was visualized directly within the gel by detecting the formation of fluorescent cyclic IDP-ribose from NHD + . The enzyme catalysed the hydrolysis of cyclic ADP-ribose to ADP-ribose. Moreover, in the presence of nicotinamide and cyclic ADP-ribose the enzyme synthesized NAD + . Both the ADP-ribosyl cyclase and NADase activities of the enzyme were strongly inhibited by reducing agents. Treatment of the NADase with dithiothreitol caused the apparent inactivation of the enzyme. Subsequent removal of the reducing agent and addition of oxidized glutathione led to a partial recovery of enzymic activity. The results support a model for pro-oxidant-induced calcium release from mitochondria involving cyclic ADP-ribose as a specific messenger, rather than the non-enzymic modification of proteins by ADP-ribose.