Properties of poly(ADP-ribose) synthetase from rat pancreas and poly(ADP-ribosylation) of basic nuclear proteins.

The isolated nuclei of rat pancreas contain an enzyme system that will incorporate 3H-labeled NAD into an acid-insoluble product, which is shown to be poly(ADP-ribose). The enzyme has an optimum pH of 7.8 and the optimum temperature is between 20 and 30 degrees C. Optimum Mg2+ concentration is 8 mM and dithiothreitol also stimulates the enzyme at a concentration of 8 mM. Under standard conditions, the Km value for the reaction is 0.25 mM and an inhibition by the substrate is observed at high substrate concentrations. It has also been found that only one basic nuclear protein, that is, histone H1, is modified by the synthetase. An average chain length of 5.0 is found in the nuclei and of 4.5 on histone H1. Radioautographic studies show that poly(ADP-ribose) is closely associated with chromatin.     

Inhibition of the transport of citrate during ADP-ribosylation of inner membrane proteins of the mitochondria

The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.     

ADP-ribosyltransferase in Plasmodium (malaria parasites).

The nuclei of Plasmodium yoelii nigeriensis contain an enzyme, ADP-ribosyltransferase, that will incorporate the ADP-ribose moiety of NAD+ into acid-insoluble product. The time, pH and temperature optima of this incorporation are 30 min, 8.5 and 25 degrees C respectively. Maximum stimulation of the enzyme activity is obtained with 1.0 mM-dithiothreitol or 2.0 mM-2-mercaptoethanol. Ca2+ and Mg2+ ions at optimum concentrations of 5 mM and 10 mM respectively stimulated the activity of the enzyme by 21% and 91%. The enzyme activity is, however, inhibited by 24% in the presence of 10 mM-MnSO4. The substrate, NAD+, exhibits an apparent Km of 500 microM, and the activity of the enzyme is inhibited by four chemical classes of inhibitors: nicotinamides, methylxanthines, thymidine and aromatic amides. The inhibitors are effective in the following increasing order: nicotinamide less than 3-aminobenzamide less than thymidine less than 5-methylnicotinamide less than theophylline less than m-methoxybenzamide less than theobromine. The enzyme activity is also inhibited by some DNA-binding anti-malarial drugs.     

ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos.

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5'-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Poly(adenosine diphosphate ribose) is covalently linked to nuclear proteins by two types of bonds.

(ADP-ribose)n residues formed by short-term incubation of adult rat liver and Ehrlich carcinoma nuclei with labeled NAD were analyzed by Cs2SO4/guanidinium chloride/urea density gradient centrifugation. Comparison with samples in which the protein had been completely digested revealed that most, or probably all, acid-insoluble (ADP-ribose)n chains are covalently bound to nuclear proteins, as is true for the short, acid-soluble (ADP-ribose)n chains. Complete release of (ADP-ribose)n chains is effected by dilute alkali. In contrast, NH2OH liberated only part of the long and the short (ADP-ribose)n residues from the protein conjugates, indicating two types of bonds, both alkali-labile, but only one susceptible to neutral hydroxylamine. Both types of bonds were equally distributed among acid-soluble and acid-insoluble (ADP-ribose)n chains. -Stability of the (ADP-ribose)n protein conjugates during isolation is only guaranteed at pH values below 7.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

Nicotinamide adenine dinucleotide glycohydrolase from rat liver nuclei. Isolation and characterization of a new enzyme.

A new type of nicotinamide adenine dinucleotide glycohydrolase (NADase) has been isolated from rat liver nuclei. When partially purified chromatin is passed through a Sephadex G-200 column in the presence of 1 M NaCl, enzyme activities catalyzing the liberation of nicotinamide from NAD elute in two peaks. One, which appears in the void volume fraction, hydrolyzes the nicotinamide-ribose linkage of NAD to produce nicotinamide and ADP-ribose in stoichiometric amounts. This activity is not inhibited by 5 mM nicotinamide. The other, which elutes much later, catalyzes the formation of poly(ADP-ribose) from NAD and is completely inhibited by 5 mM nicotinamide. The former, NADase, is DNase-insensitive and thermostable, has a pH optimum of 6.5 to 7, a Km for NAD of 28 muM, and a Ki for nicotinamide of 80 mM, and hydrolyzes NADP as well as NAD. The latter, poly(ADP-ribose) synthetase, is sensitive to DNase treatment and heat labile, has a pH optimum of 8 to 8.5, a Km for NAD of 250 muM and a Ki for nicotinamide of 0.5 mM and is strictly specific for NAD. Further, the former NADase is shown to lack transglycosidase activity, which has been documented to be a general property of NADases derived from animal tissues. These results indicate that the NAD-hydrolyzing enzyme newly isolated from nuclei is a novel type of mammalian NADase which catalyzes the hydrolytic cleavage of the nicotinamide-ribose linkage of NAD.     

Effects of hyperthermia and nicotinamide on DNA repair synthesis, ADP-ribosyl transferase activity, NAD+ and ATP pools, and cytotoxicity in gamma-irradiated human mononuclear leukocytes

Effects of hyperthermia and nicotinamide on ADP-ribosyl transferase activity (ADPRT), unscheduled DNA synthesis (UDS), NAD+- and ATP-pools and cytotoxicity were investigated in gamma-irradiated human mononuclear leukocytes. A significant decrease in radiation-induced UDS after heat treatment for 45 min was found. Nicotinamide increased the UDS levels in irradiated cells, but no effect of hyperthermia on these increased UDS values was observed. In the presence of 2 mM nicotinamide radiation-induced ADPRT activity was reduced to about 50 per cent. However, hyperthermia for 45 min was found to have no effect on the enzyme activity for temperatures below 46 degrees C. Nicotinamide increased the NAD+ pool in unirradiated cells. Damaging the cells with gamma-radiation leads to a severe depletion of the NAD+ pool. The NAD+ pool is restored, however, if the cells repair for 5 h at 37 degrees C. When radiation-damaged cells were treated with hyperthermia, exogenously supplied nicotinamide could not be converted to NAD+ in sufficient amounts to prevent NAD+ depletion. These data indicate that the radiosensitizing effect of heat and nicotinamide could both be explained by effects on the enzyme ADPRT, i.e. nicotinamide by directly blocking the enzyme and hyperthermia by limiting the co-substrate (NAD+).     

Dependence of ADP-ribosylation of histones of chicken liver nuclei on the intranuclear content of NAD

The dependence of ADP-ribosylation of chicken liver nuclear histones on NAD concentration in the nuclei was studied under conditions of stimulation of coenzyme synthesis by the nicotinamide and nicotinic acid as well as upon addition of various concentrations of the [Ado-U-14C]NAD nuclei to the incubation mixture. In the first case, the rate of [Ado-U-14C]NAD incorporation into the histones was decreased due to the dilution of the label by the de novo synthesized NAD. The amount of the latter formed under effects of nicotinic acid and nicotinamide increased, correspondingly, from 2,2 X 10(-5) mmol up to 4.1 X 10(-5) and 7.0 X 10(-5) mmol per mg of nuclear protein. The incorporation of [Ado-U-14C]NAD into the histones decreased from 12.0 X 10(-8) mmol after incubation of liver slides with nicotinic acid and nicotinamide down to 8.0 X 10(-8) and 7.0 X 10(-8) mmol, respectively. With a rise in the concentration of exogenous [Ado-U-14C]NAD, the level of ADP-ribosylation of nuclear histones increased, the plot [14C]NAD incorporation at the labeled coenzyme concentration of 25 X 10(-7) mM/mg of histone had a plateau. Changes in the labeled substrate concentration brought about corresponding changes in the average length of the histone-linked poly-(ADP-ribose) chain.     

Mono(ADP-ribosylation) in rat liver mitochondria

This paper investigates protein mono(ADP-ribosylation) in rat liver mitochondria. In isolated inner mitochondrial membranes, in the presence of both ADP-ribose and NAD+, a protein is mono-(ADP-ribosylated) with high specificity. The reaction apparently consists of enzymatic NAD+ glycohydrolysis and subsequent binding of free ADP-ribose to the acceptor protein. In terms of chemical stability, the resulting bond is unique among the ADP-ribose linkages thus far characterized. Formation of a Schiff base adduct between free ADP-ribose and the acceptor protein is excluded. In intact mitochondria at least three classes of proteins are ADP-ribosylated in vivo. One ADP-ribose-protein linkage is of the carboxylate ester type as indicated by its lability in neutral buffer. Another class of ADP-ribosylated proteins requires hydroxylamine for release of ADP-ribose. The third class is stable in hydroxylamine but labile to alkali, similar to the ADP-ribose-cysteine linkage in transducin formed by pertussis toxin.     

Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was considered to be because even at 50 degrees C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.     

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Br?nsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.     

Poly(adenosine diphosphate ribose) polymerase in Physarum polycephalum.

1. The isolated nuclei of the slime mould Physarum polycephalum contain an enzyme that will incorporated [adenine-3H] NAD+ into an acid-insoluble product, which is shown to be poly(ADP-ribose). 2. This incorporation has an optimum pH of 8.2 and a temperature optimum below 10degreesC. 3. Optimum stimulation is given by 15 mM-Mg2+. 4. 2-Mercaptoethanol or dithiothreitol also stimulates the incorporation, the latter at an optimum concentration of about 1 mM. 5. Under optimum conditions the Km value for the reaction is 0.28 mM at 15degreesC. Nicotinamide inhibits the incorporation with a Ki of 5.7 muM. 6. Exogenous DNA stimulates the incorporation by about 100%. 7. Preincubation of the nuclei with deoxyribonuclease, but not with ribonuclease, almost completely inactivates the incorporation of NAD+. 8. The enzyme is unstable at both 0degrees and 15degreesC in the absence of dithiothreitol. The presence of dithiothreitol at a concentration of 1 mM stabilizes the enzyme at both these temperatures. 9. The activity of this enzyme per nucleus was shown in three separate experiments to fall by about one-half in early S phase and then to rise to its pre-mitotic value after about 3 h, that is in late S phase. 10. The possible physiological function of this enzyme system is discussed.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Small molecule regulation of Sir2 protein deacetylases

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.     

Pyridine nucleotide cycling and poly(ADP-ribose) synthesis in resting human lymphocytes

NAD is a critical cofactor for the oxidation of fuel molecules. The exposure of human PBL to agents that cause DNA strand breaks to accumulate can deplete NAD pools by increasing NAD consumption for poly(ADP-ribose) formation. However, the pathways of NAD synthesis and degradation in viable PBL have not been carefully documented. The present experiments have used radioactive labeling techniques to trace the routes of NAD metabolism in resting PBL. The cells could generate NAD from either nicotinamide or nicotinic acid. PBL incubated with [14C]nicotinic acid excreted [14C]nicotinamide into the medium. Approximately 50% of a prelabeled [14C]NAD pool was metabolized during 6 to 8 hr in tissue culture. Basal NAD turnover was prolonged threefold to fourfold by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) synthetase. Supplementation of the medium with 3-ABA also prevented the accelerated NAD degradation that ensued after exposure of PBL to deoxyadenosine plus deoxycoformycin at concentrations previously shown to cause DNA strand break accumulation. These results demonstrate that quiescent human PBL continually produce NAD and utilize the nucleotide for poly(ADP-ribose) synthesis.