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FEBS Letters 585 (2011) 1651–1656

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Review Compartmentation of NAD+-dependent signalling ⇑ ⇑ Friedrich Koch-Nolte a, , Stefan Fischer b, Friedrich Haag a, Mathias Ziegler b,

a Institute of Immunology, University Medical Center, Martinistr. 52, 20246 Hamburg, Germany b Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5008 Bergen, Norway

article info abstract

Article history: NAD+ plays central roles in energy metabolism as redox carrier. Recent research has identified Received 25 February 2011 important signalling functions of NAD+ that involve its consumption. Although NAD+ is synthesized Revised 21 March 2011 mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracel- Accepted 21 March 2011 lular compartments. Three families that consume NAD+ in signalling reactions have been Available online 31 March 2011 characterized on a molecular level: ADP-ribosyltransferases (ARTs), (SIRTs), and NAD+ glyco- Edited by Sergio Papa, Gianfranco Gilardi (NADases). Members of these families serve important regulatory functions in various and Wilhelm Just cellular compartments, e.g., by linking the cellular energy state to expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracel- + Keywords: lular compartment. Distinct NAD pools may be crucial for these processes. Here, we review the cur- + NAD+ rent knowledge about the compartmentation and biochemistry of NAD -converting that + ADP-ribosyltransferase control NAD signalling. Sirtuins Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. NAD+ glycohydrolase NAD+ signalling

1. Introduction cuses on the biochemistry and compartmentation of NAD+-con- verting enzymes and of NAD+ itself in mammalian cells. The variety of physiological roles played by NAD+ has long been underestimated. Besides its vital functions as electron carrier, this 2. Conversions of NAD+ in signalling reactions pyridine nucleotide has important functions in signalling pathways [1–4]. The expression of several is linked to the redox state of In redox reactions, NAD+ is reversibly converted to NADH by + pyridine nucleotides via NAD /NADH-binding that act as addition of two electrons and a proton to the ring, + redox sensors [5,6]. NAD also mediates post-translational protein thereby oxidizing metabolic intermediates (Fig. 1). In contrast, + modifications such as ADP-ribosylation and NAD -dependent pro- NAD+-dependent signalling reactions involve cleavage of the glyco- tein deacetylation [7–13]. Moreover, it is a direct precursor of mes- sidic bond between nicotinamide and ADP-ribose (wavy red line in senger molecules such as ADP-ribose (ADPR), cyclic ADP-ribose Fig. 1), i.e. the consumption of NAD+. These reactions result in the (cADPR) and O-acetyl-ADP-ribose (OAADPR), which are all in- transfer of the ADP-ribose moiety onto an acceptor, and release of 2+ + volved in intracellular Ca mobilization [14–16]. NAD -mediated the remaining nicotinamide. Therefore, these reactions are also re- signalling has now moved into focus as it governs fundamental ferred to as ADP-ribosyl transfer. biological processes both on a cellular and organismal level. To ful- fil such a broad range of functions a variety of mechanisms leading 2.1. NAD+-dependent protein modification and generation of second + to signalling conversions of NAD have evolved. Several enzymes messengers have been identified that catalyse different conversions of NAD+ leading to highly specific or rather general physiological conse- There are two principal types of NAD+-dependent signalling quences. This multitude of different signalling pathways requires reactions, the modification of proteins and the generation of mes- precise spatial organization. Several excellent recent reviews have senger molecules (Fig. 1). NAD+-dependent protein modifications covered various aspects of NAD signalling [1–16]. This review fo- include ADP-ribosylation reactions, that is, covalent attachment of ADP-ribose to specific amino acid residues, and the deacetyla- tion of residues, catalysed by ADP-ribosyltransferases (ARTs) and sirtuins (SIRTs), respectively. ADP-ribosylation and deacetyla- ⇑ Corresponding authors. Fax: 4940 7410 54243 (F. Koch-Nolte). tion activate or inactivate target protein functions, as in the case of E-mail addresses: [email protected] (F. Koch-Nolte), [email protected] (M. Ziegler). other posttranslational protein modifications. ADP-ribosylation is a

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.03.045 1652 F. Koch-Nolte et al. / FEBS Letters 585 (2011) 1651–1656

Fig. 1. NAD+-converting enzymes and NAD+-dependent signalling. In redox reactions, NAD+ is reversibly converted to NADH by addition of two electrons and a proton to the nicotinamide ring. NAD+-converting enzymes cleave the glycosidic bond in NAD+ linking nicotinamide to ribose (indicated by the red wave). ADP-ribosyltransferases (ART) transfer ADPR onto amino acid residues (mono-ADP-ribosylation) or onto protein-linked ADP-ribose moieties (poly ADP-ribosylation). These protein modifications are reversed by ADP-ribosylhydrolases (ARH) and poly-ADP-ribosyl-glycohydrolase (PARG), liberating ADPR. Sirtuins (SIRT) catalyse NAD+-dependent deacetylation of proteins carrying acetylated lysine residues, yielding O-acetyl-ADPR (OAADPR). NAD-glycohydrolases (NADase) catalyse hydrolysis of NAD+ to ADPR and nicotinamide and cyclisation of ADPR to cyclic-ADPR. NADases also catalyse substitution of the nicotinamide moiety in NADP+ by nicotinic acid, yielding NAADP. These NAD+-derivatives can trigger elevation of intracellular Ca2+. reversible protein modification. Based on the extent of the modifi- linked ADP-ribose moieties, generating polymers of ADP-ribose cation, two modes can be distinguished, mono-ADP-ribosylation (PARsylation) [11]. One member of the ART family catalyses trans- and poly-ADP-ribosylation. ADP-ribose is released from mono- or fer of ADP-ribose to the terminal phosphate in t-RNA, initiating the poly-ADP-ribosylated proteins by ADP-ribosylhydrolases (ARHs) splicing reaction and the release of ADP-ribose 10–20 cyclic phos- and poly-ADP-ribose glycohydrolase (PARG) [10]. NAD+-dependent phate [22,23]. The ART fold has been found in proteins from all protein deacetylation involves transfer of the from the kingdoms of life. In , the ART family is the largest and, target protein onto ADP-ribose resulting in the formation of O- perhaps, most versatile of the three NAD+-converting protein fam- acetylated ADP-ribose [12]. Both ADPR and OAADPR have been ilies [10]. The 20–25 members of the ART family can be subdivided implicated as second messengers. Second messengers can also be into two structurally distinct subclasses designated ARTC and generated from NAD+ by ADP-ribosylcyclases/NAD+ glycohydrolas- ARTD on the basis of structural similarities to cholera and diphthe- es (NADases), yielding ADPR and cADPR [14]. NADases also cata- ria toxins, respectively [11]. Members of the smaller ARTC subfam- lyse the substitution of the nicotinamide moiety by nicotinic acid ily are expressed as GPI-anchored or secreted ecto-enzymes. in NADP (generated from NAD+ by NAD kinase), thereby forming Members of the larger ARTD subfamily include nuclear poly- nicotinic acid adenine dinucleotide phosphate (NAADP). All these ADP-ribosyltransferases, the t-RNA dephosophorylating NAD+-derived second messengers contribute to intracellular cal- TpT, and cytosolic oligo- and mono-ADP-ribosyltransferases. cium signalling by acting on calcium channels in the plasma mem- SIRTs have a low intrinsic NAD+ binding affinity, and NAD+- brane, endoplasmatic reticulum or lysosomes. binding to SIRTs generally requires prior binding of an acetylated peptide [18,19]. This peptide forms the central strand of a three- 2.2. Three NAD+-converting protein families stranded enzyme-substrate b-sheet in a crevice between the small Zn-binding domain and the large Rossman nucleotide-binding fold. In enzymes catalysing redox reactions the adenine and pyridine NAD+ binds in an extended conformation that exposes the alpha moieties of NAD+/NADH typically each are bound by a separate face of the nicotinamide ribose to the carbonyl oxygen of the acetyl classic Rossmann/Walker nucleotide-binding fold. The CtBP family lysine substrate promoting formation of an O-alkylamidate reac- of redox-responsive factors similarly binds NAD+/ tion intermediate, the deacetylation of lysine and the release of NADH with two separate Rossman fold [17]. The enzymes catalys- O-acetyl ADP-ribose. The SIRT fold has been found in proteins from ing NAD+-consumption fall into three individual protein families all kingdoms of life [24,25]. The seven members of the mammalian containing distinct NAD+-binding domains. SIRTs use a single copy family are located in the nucleus, cytosol, and mitochondria of the Rossman fold for coordinating the adenine moiety of NAD+ [24]. [18,19]. ARTs and NADases carry rather distinct NAD+-binding NADases bind NAD+ in a bent conformation [26,27]. These en- domains. zymes facilitate base-exchange of the nicotinamide moiety, intra- ARTs bind NAD+ in an extended conformation in a Pacman-like molecular cyclisation or hydrolysis of the high energy bond. The crevice formed by two abutting b-sheets [20,21]. Nicotinamide is NADase fold has been identified so far only in proteins of metazoan buried in a deep pocket, exposing the glycosidic bond between nic- animals [16]. In mammals, the two members of the ADP-ribosylcy- otinamide and the adjacent ribose moiety for nucleophilic attack clase/NADase family are expressed as membrane bound ecto- by a variety of substrates, including amino acids, nucleotides, enzymes [16,28]. ADP-ribose, phosphate, antibiotics, and water. Mammals use the Some members of these protein families show overlapping ART family mainly for posttranslational ADP-ribosylation of amino enzyme activities, e.g., hydrolysis of NAD+ by some ARTs and the acid residues in proteins and for the ADP-ribosylation of protein- ADP-ribosylation of proteins by some SIRTs. F. Koch-Nolte et al. / FEBS Letters 585 (2011) 1651–1656 1653

2.3. Physiological functions of NAD+-dependent signalling processes Second messenger molecules generated during NAD+-depen- dent protein modifications or directly from NAD+ or NADP+ by Over the last years, important discoveries have been made with NADases trigger the release of calcium from intracellular stores regard to all the signalling conversions mediated by NAD+. Never- or entry of calcium through plasma membrane channels [14,15]. theless, NAD+-dependent protein deacetylation by SIRT1 has run Cyclic ADP-ribose activates the ryanodine receptor leading to the the show lately, because its activity has been associated with a release of calcium from the endoplasmic reticulum, similar to ino- number of fundamental biological processes, most prominently sitol-1,4,5-trisphosphate (IP3), which activates the IP3 receptor. In regulation of the circadian and ageing [12,24,25]. SIRT1 addition, cADPR is a ligand of the TRPM2 channel (a member of the deacetylates histones at specific gene loci and thereby silences transient receptor potential cation channel family) and thereby expression of the corresponding genes. Accordingly, activation of stimulates the influx of calcium from the extracellular space. Both the SIRT1 homologues in non-mammalian species such as yeast, ADPR and OAADPR have also been reported to activate TRPM2 or flies leads to substantial extension of life channels. NAADP releases calcium from intracellular, possibly cell span. In mammals, SIRT1 has been shown to regulate the activity of type-specific stores by activating lysosomal calcium channels and important transcription factors such as , PGC1a, Forkhead-box the ryanodine receptor [32–35]. The capacity of NAD+-derived proteins and others. Thereby, its activity affects metabolic homeo- messengers, in addition to IP3, to selectively trigger calcium re- stasis, participates in the regulation of the biological clock, and in- lease into the cytosol may provide mechanisms to confine calcium creases stress resistance. Moreover, sirtuins regulate the activity of signalling in time and space and to allow calcium signalling by spe- key metabolic enzymes. In most cases, the deacetylation leads to cific mechanisms in response to different extracellular signals. catalytic activation of the target proteins. The ART family also has a broad scope of regulatory functions, ranging from DNA repair to sensing cell death in inflammation 3. Compartmentation of NAD+-dependent signalling pathways [1,10,11]. Poly-ADP-ribosylation has largely been associated with nuclear events [9]. The most prominent enzyme, referred to as Given the variety of NAD+-degrading processes and subsets of poly-ADP-ribose polymerase 1 (PARP1) or ARTD1 [11], has a criti- enzymes competing for the same substrate, NAD+-dependent reg- cal function in DNA repair, based on its high affinity to DNA lesions. ulatory pathways need to be tightly regulated, e.g., by specific up- Under normal physiological conditions PARP1 is rather inactive, stream mechanisms that govern the activation or inhibition of whereas its binding to DNA strand breaks dramatically increases individual NAD+-dependent mechanisms [3,4]. Moreover, NAD+- its catalytic activity. Thereby, the site of DNA damage is ‘‘marked’’ dependent signalling pathways are compartmentalized, providing with polymers of ADP-ribose, which facilitates the recruitment of another level of regulation and sophistication. Fig. 2 depicts the DNA repair factors. PARP1 also participates in the regulation of cellular distribution of known enzyme activities contributing to transcription, the regulation of structure and other nu- NAD+-dependent regulatory pathways and illustrates estimated clear processes related to DNA rearrangements [9,29]. Following concentrations of NAD+ in different cellular compartments [4]. the release of NAD+ from cells during inflammation, ARTs tethered Interestingly, a number of NAD+-dependent regulatory reactions to the extracellular leaflet of the plasma membrane ADP-ribosylate take place on the outer surface of the plasma membrane, despite the ectodomains of numerous cell surface proteins, and secretory the rather low concentration of NAD+ (submicromolar) in extracel- ARTs ADP-ribosylate inflammatory cytokines [8]. These extracellu- lular fluids, e.g., serum [2]. Below, we provide a few examples of lar ADP-ribosylation reactions play important regulatory roles in NAD+-mediated signalling processes that take place in different cellular communication and immune cell homeostasis [30,31]. subcellular compartments and on the cell surface.

Fig. 2. Compartmentation of NAD+ and NAD+-converting enzymes. In mammals, NAD+-converting enzymes have been identified in the extracellular space, the cytosol, the nucleus and mitochondria. The concentration of NAD+ is high in mitochondria (400 lM), intermediate in the nucleus and cytosol (100 lM) and low (<1 lM) in the extracellular space, and can vary considerably depending on the cell type, metabolic condition, stress, and redox status. Mounting evidence indicates that vesicular compartments including the endoplasmic reticulum, the Golgi apparatus, exocytotic vesicles and peroxisomes also harbour NAD+, albeit at concentrations that are extremely difficult to assess experimentally (indicated in grey). 1654 F. Koch-Nolte et al. / FEBS Letters 585 (2011) 1651–1656

3.1. Extracellular NAD+-dependent signalling GPCRs expressed by smooth muscle cells in blood vessels, the kid- ney and the gut [48,49]. The plasma membrane is impermeable to NAD+. However, NAD+ is released from cells by lytic and non-lytic mechanisms following 3.2. Nuclear NAD+-dependent signalling disruption of membrane integrity by pore forming proteins, mechanical injury or osmotic forces [30,36]. Unless repaired, large Prominent NAD+-dependent processes in the nucleus include or enduring leaks in the cell membrane cause breakdown of the poly-ADP-ribosylation and protein deacetylation. Besides PARP1 electrochemical potential and cell death. The leakage of NAD+ from (ARTD1), other ARTs generating protein-bound PAR play important dying cells may represent an ancient danger signal and a universal roles. PARP2 (ARTD2), even though catalytically less active, appears ‘‘molecular odorant’’ of dead cells. Lysis of cells in multicellular to facilitate DNA repair, similar to, or in concert with PARP1. Poly- organisms often accompanies infections by microbial pathogens, ADP-ribosylation is also involved in the regulation of telomerase e.g., during infection of cells with a lytic virus, puncture of the cell activity. The telomeric repeat binding factor 1 (TRF1) prevents ac- membrane by bacterial haemolysins and other microbial or endog- cess of telomerase to telomers by binding to the telomeric DNA. enous pore-forming proteins, e.g., complement factors or perforins When activated, the ART enzyme tankyrase 1 (ARTD5) catalyses secreted by cytotoxic T cells and natural killer cells. It is perhaps poly-ADP-ribosylation of TRF1. Thereby, TRF1 loses its DNA bind- not surprising, then, that metazoan immune cells have developed ing ability and telomerase gains access to extend telomers. PARP1 sensors for NAD+ and its metabolites in the form of ecto-enzymes, can modulate transcription in a similar fashion by modifying spe- ligand-gated ion channels, and G-protein-coupled receptors cific transcription factors that become unable to bind to their cog- (GPCRs) [8,28,37]. nate sites in the DNA. PARG activity in the nucleus rapidly Several members of the ART family are expressed on the outer degrades PAR, making poly-ADP-ribosylation a rather transient leaflet of the plasma membrane as GPI-anchored ecto-enzymes modification. or as secretory proteins. In response to NAD+ released from cells, The best studied nuclear sirtuin is SIRT1. Numerous studies these enzymes ADP-ribosylate other cell surface and secretory pro- have documented that SIRT1 deacetylates not only histones, but teins. This provides a mechanism of translating pulses of leaked also critical transcription factors. Among the consequences of NAD+ into longer-lasting imprints [31,36]. The ADP-ribosylation SIRT1-mediated transcriptional regulation is the favourable adjust- of human neutrophil peptide 1 on Arg14 and Arg24 by ARTC1 ment of energy metabolism. In this regard, among the major tar- and the ADP-ribosylation of the P2X7 ion channel on Arg125 and gets of SIRT1 deacetylation are PGC1a, PPARc and FOXO Arg133 by ARTC2 provide well-characterized examples of cell sur- transcription factors. Therapeutic concepts, based on SIRT1 activa- face and secretory proteins regulated by ADP-ribosylation [8,38– tion, begin to emerge which are designed to counteract, for exam- 40]. ple, diet-induced obesity, obesity-induced diabetes and metabolic NAD+ hydrolysis by CD38, a member of the NADase family teth- syndrome [50]. Another example as to how SIRT1 activity modu- ered to the cell surface, is an important mechanism to limit the lates the function of an organism as a whole was provided by its duration of NAD+-signalling in the extracellular compartment identification as an important regulator of the biological clock. It [41]. Moreover, this enzyme generates potent calcium-mobilizing has been proposed that, at least in metabolically active tissues, second messengers, e.g., ADP-ribose and cyclic ADP-ribose. SIRT1 may act as a ‘‘rheostat’’ within the clock system and deter- Whether and how these messengers enter the cells to elevate the mine the epigenetic regulation of the clock system through cytosolic calcium remains unknown. However, on the cell surface NAD+-dependent deacetylation [51]. they are also further catabolised by extracellular pyrophospha- SIRT2 has been observed to reside both in the nucleus and the tase/phosphodiesterases (ENPP, CD203) and 50 nucleotidase (ENT, cytosol. Besides histones, it targets p300, a crucial protein acety- CD73) [42], thereby generating AMP and adenosine, which can lase. Interestingly, SIRT2 and p300 interact reciprocally. That is, activate members of the P2Y and P1 subfamilies of GPCRs. The p300 acetylates SIRT2, while SIRT2 deacetylates p300. metabolites adenosine, nicotinamide, and phosphate can be taken up by cells and used for the regeneration of NAD+ [42]. 3.3. Cytosolic NAD+-dependent signalling Dying cells also release cytosolic enzymes. Serum levels of cre- atine kinase and amino-acid , for example are used as ADP-ribosylation of elongation factor 2, actin, rho, and Ga, cat- indicators of muscle and liver damage, respectively. Some cytosolic alysed by bacterial toxins that translocate into the cytosol of mam- enzymes involved in NAD+-signalling have also been detected in malian cells, profoundly perturb cellular functions, causing severe serum: serum levels of (ADA), the enzyme diseases including diphtheria, whooping cough and cholera catalysing conversion of adenosine into inosine, increase in rheu- [52,53]. These toxins may mimic endogenous regulatory processes, matoid arthritis and other chronic inflammatory diseases [43]. and biochemical studies, indeed, have detected similar enzyme NAMPT, the enzyme catalysing the rate-limiting step in NAD+ syn- activities in various tissues [54]. However, the corresponding cyto- thesis, reportedly functions also as a cytokine acting on B cells and solic proteins have so far eluded molecular characterisation. An- neutrophils [44,45] and as an adipocytokine [46]. It is conceivable, other intriguing example of cytosolic ADP-ribosylation is the though not established, that other intracellular enzymes involved activity of vault-PARP (ARTD6): this enzyme catalyses reversible in NAD+-signalling might be released from cells in enzymatically oligo-ADP-ribosylation of the major vault protein, the main struc- active form. However, the pathophysiological interplay of bona- tural component of a huge cytosolic ribonucleoprotein complex fide extracellular NAD+-metabolising enzymes such as ART2 and thought to be involved in mRNA transport [55]. Characterisation CD38 and released cytosolic enzymes such as NAMPT and ADA still of cytosolic ADP-ribosylation reactions is hampered by the ubiqui- need to be explored. tous presence of cytosolic enzymes (ARH1 and PARG) that effi- Apart from the lytic release of NAD+ from injured or dying cells, ciently reverse mono- and poly-ADP-ribosylation. mounting evidence indicates that NAD+ can also be released from SIRT2 regulates adipocyte differentiation by deacetylation of cells in a regulated fashion, e.g., by reversible opening of pores FOXO1 in the cytosol [56]. Moreover, it was identified as a tubulin formed by members of the gap-junction , e.g., Conn- deacetylase [57] and thereby contributes to the regulation of exin 43 and Pannexin 1, or by vesicular exocytosis [47]. NAD+ microtubule-associated functions. reportedly is released from motor nerve terminals upon electrical An unresolved problem has been the generation of NAD+/ stimulation and may act as a neurotransmitter on purinergic NADP+-derived calcium messengers in the cytosol. All these mes- F. Koch-Nolte et al. / FEBS Letters 585 (2011) 1651–1656 1655 sengers (ADPR, cADPR, NAADP, and OAADPR) trigger the elevation from the cytosol. NAD+ exchange over the inner mitochondrial of cytosolic calcium and are known to act at the cytosolic side of membrane has been discovered in plant and yeast mitochondria calcium channels present in organellar or the plasma membranes. [61,62], but not in animals. While ADPR and OAADPR originate from reversible ADP-ribosyla- Even though conclusive experimental evidence is lacking (and tion or NAD+-dependent protein deacetylation, respectively, the very hard to obtain), the cytosolic and nuclear NAD+ pools are most pathway of how cADPR and NAADP arise in the cytosol has re- often considered to be readily exchangeable. Therefore, it is gener- mained unclear. Both known mammalian NADases, CD38 and ally assumed that the nuclear and cytosolic NAD+ concentrations CD157, are ecto-enzymes and no other candidate enzyme that are similar. Whether these are valid suppositions remains to be might catalyse the synthesis of these messengers in the cytosol seen. Interestingly, the known subcellular distribution of NAD+- has been identified. Therefore, transport mechanisms have been dependent signalling mechanisms coincides with the established invoked which could mediate, for example, the entry of cADPR presence of NAD+ in these compartments. The biochemical func- from the extracellular space or through the membranes of endo- tions of peroxisomes (e.g., fatty acid oxidation) also imply the pres- somes following internalization of CD38. Irrespective of these ence of NAD+ in these organelles. Indeed, in a recent report the uncertainties, cytosolic calcium signalling mediated by NAD+/ presence of NAD+ in peroxisomes was visualized [63]. Surprisingly, NADP-derived messengers has been intensely studied and demon- this study also established that the endoplasmic reticulum and the strated in a multitude of model systems: Secretory functions of Golgi complex constitute NAD+ pools that are likely independent of pancreatic acinar cells are coordinated by NAADP and cADPR, T cell the cytosolic one. However, so far there is no information regarding activation relies on successive cytosolic calcium elevations trig- the NAD+ concentration in these compartments (as indicated by gered by different messengers, and in the brain, the regulation of their grey shading in Fig. 2), nor whether their NAD+ pools also par- TRPM2 channels by NAD+-derived ligands is crucial. ticipate in regulatory processes. The versatility of NAD+ conversions in the extracellular space is 3.4. Mitochondrial NAD+-dependent signalling in apparent contradiction to the rather low NAD+ concentration measured in extracellular fluids (e.g., submicromolar in plasma, While mitochondria had been largely regarded as purely meta- see Fig. 2). It is conceivable that the overall concentration may re- bolic and bioenergetic workhorses, research over the last decades flect an average of high local concentrations and an otherwise identified these organelles as key players in central regulatory nearly complete absence of NAD+ in extracellular fluids. That is, mechanisms. It is now clear that NAD+-mediated signalling within spatially confined release of NAD+ may have paracrine function these organelles plays a fundamental role in metabolic regulation. or signal cell damage to alert repair systems. Key components of the respiratory chain, fatty acid degradation, and ammonia detoxification are regulated by the three mitochon- 5. Perspectives drial sirtuins, SIRT3, SIRT4, and SIRT5 [13]. SIRT3 has been best studied and was shown to deacetylate a number of mitochondrial NAD+ has emerged as an important mediator of signalling both proteins including enzymes of the b-oxidation pathway, glutamate between and within cells. This nucleotide undergoes a variety of dehydrogenase, and a subunit of complex I in the respiratory chain. conversions leading to protein modifications or activation of ion SIRT5 accelerates the urea cycle by deacetylating carbamoylphos- channels with pronounced physiological effects both on the cellu- phate synthase 1, the rate-limiting enzyme [58]. Besides acetyla- lar and organismal level. The consumption of NAD+ by different en- tion/deacetylation, glutamate dehydrogenase also appears to be zymes necessitates continuous resynthesis of the molecule. How regulated by mono-ADP-ribosylation mediated by SIRT4 [59]. NAD synthesis and consumption are affected by the subcellular While the ADP-ribosylation is inhibitory, deacetylation apparently localization of the enzymes participating in NAD+-synthesis and releases the inhibition caused by the acetylation of enzymes [13]. NAD+-conversion, and how the supply of NAD+ to different cellular Interestingly, the synthesis of acetyl-CoA, the substrate for protein compartments is organized remain challenging questions. New re- acetylation, is regulated by acetylation/deacetylation of acetyl-CoA search tools including specific antibodies, agonists, and antagonists synthase (ACS). Both the cytosolic and the mitochondrial isoforms directed against the key enzymes of NAD+ synthesis and consump- + are activated by NAD -dependent deacetylation [60]. tion will help address these questions. Moreover, these reagents may pave the way to a new generation of therapeutics for treating + + 4. NAD pools and NAD -dependent signalling metabolic and inflammatory diseases in which NAD+ signalling is impaired. 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