Energy and signal transduction by NAD:
Signalling pathways and subcellular
compartmentation of NAD biosynthesis

Mathias Ziegler

UoB

Science
    Faculty

MBI

Research
- Fjose
- Fladmark
- Goksøyr
- Helland
- Jensen
- Lillehaug
- Male
- Raae
- Seo
- Ziegler
- Walther
- Aasland

Since the beginning of the last century, seminal discoveries have identified pyridine nucleotides as the major redox carriers in all organisms. Recent research has unravelled an unexpectedly wide array of signalling pathways that involve nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP. NAD serves as substrate for protein modification including protein deacetylation, and mono- and poly-ADP-ribosylation. Both NAD and NADP represent precursors of intracellular calcium-mobilizing molecules. It is now beyond doubt that NAD(P)-mediated signal transduction does not merely regulate metabolic pathways, but might hold a key position in the control of fundamental cellular processes. The comprehensive molecular characterization of NAD biosynthetic pathways over the past few years has further extended the understanding of the multiple roles of pyridine nucleotides in cell biology.

Energetic Functions of NAD(P)
Being the major redox carriers in metabolic reactions the pyridine nucleotides (NAD and NADP) are essential molecules in all organisms. Although most dehydrogenases accept both NAD and NADP, there is a preference for NAD+ in oxidative, catabolic routes. On the other hand, NADPH is preferentially used for reductive syntheses catalysed by substrate-specific reductases. In catabolic reactions NAD+ receives two electrons and a proton from a substrate molecule. A second proton of the substrate is released. More: NAD in Bioenergetics

Signalling Functions of Pyridine Nucleotides
Besides their well-established roles in energy transduction, NAD and NADP also hold a key position in a variety of regulatory pathways. Similarly to such a dual function of ATP, NAD(P)+ is used for covalent protein modification and the generation of biologically active messenger molecules. The reactions of NAD+-mediated protein modification include mono- and poly-ADP-ribosylation as well as NAD+-dependent protein deacetylation. Moreover, both NAD+ and NADP+ serve as precursors of intracellular calcium mobilising agents including cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP).

Poly-ADP-ribosylation is activated by DNA strand breaks. Therefore, agents causing DNA damage (e.g., reactive oxygen species) can also trigger massive consumption of NAD. Apparently, the loss of NAD may be a critical factor for cell survival. Consequently, the capacity to resynthesize NAD is an importantÊ cellular function.

NAD(P) Biosynthesis
Considering the absence of degradation in the redox reactions, the need for continuous NAD(P) synthesis had been considered minimal over many years. Nevertheless, the routes of NAD(P) generation were established long ago. However, until recently, a molecular characterisation of the enzymes of NAD(P) biosynthesis was lacking. Owing to the discovery of a variety of NAD-mediated regulatory pathways involving the cleavage of NAD, the molecular analysis of NAD(P) biosynthesis has now become a topic of major interest. In particular, as pointed out above, poly-ADP-ribosylation may consume large amounts of NAD, primarily in situations of cellular stress. In some instances the cellular NAD may be almost depleted. Therefore, only if there is a highly efficient mechanism of NAD resynthesis, the cells will be able to survive such conditions. In addition, it has been demonstrated that manipulation of biosynthetic pathways of NAD may influence fundamental cellular processes such as gene regulation and ageing.

Biosynthetic Pathways in Mammalians
Nicotinamide serves as the major precursor of NAD in mammalians. The reaction with phosphoribosyl pyrophosphate (PRPP) leads to the formation of nicotinamide mononucleotide, NMN. Finally, this mononucleotide is linked to the adenylate moiety of ATP forming NAD. This reaction is catalysed by nicotinamide mononucleotide adenylyltransferases, NMNATs. As an alternative to nicotinamide, quinolinic acid (a degradation product of tryptophan) may be used to form nicotinic acid mononucleotide (NaMN). All known NMNATs may use NaMN to form a dinucleotide and the subsequent reaction with ATP then yields nicotinic acid adenine dinucleotide, NAAD. This intermediate is the substrate of NAD synthase, an enzyme which amidates NAAD to NAD using glutamine (instead of free ammonium, as found in prokaryotes) as source of ammonium. The phosphorylation of NAD at the 2' position yields NADP and is catalysed by NAD kinase.

Regulatory Function of NAD Metabolism
A major influence of NAD metabolism on lifespan regulation was originally found in yeast. Histone deacetylation by Sir2p results in gene silencing and thereby in extended lifespan. It has been found that nicotinamide is a potent endogenous inhibitor of Sir2 enzymes (sirtuins). Therefore, speeding up the reaction metabolising nicotinamide will diminish the cellular concentration of this Sir2p inhibitor. Even the acceleration of a subsequent reaction in the pathway (e.g., overexpression of NMNAT) could enhance nicotinamide utilisation by shifting the equilibrium and thereby increase Sir2p activity. Indeed, overexpression of enzymes of the NAD biosynthetic or salvage pathways has been demonstrated to extend lifespan, presumably by releasing the inhibition of Sir2p.However, it should be borne in mind that other enzymes using NAD in regulatory reactions, including PARPs and ADP-ribosyl cyclase, are also inhibited by nicotinamide. In fact, nicotinamide has been used as PARP inhibitor in a variety of experimental settings. Perhaps, some of these studies need to be re-evaluated with regard to effects of nicotinamide on other important regulatory mechanisms such as NAD-dependent protein deacetylation.

Subcellular Compartmentation of NAD and Its Metabolism
The cellular NAD concentration has been measured many times in a variety of tissues and cells and can generally be stated to be in the low (sub)millimolar range. It has to be noted, however, that a considerable amount of the dinucleotide is protein-bound in vivo. Additional variablity has been observed with regard to the ratio of NAD+/NADH, even at resting state. Clearly, this ratio also depends on the metabolic conditions.

Compartmentation of NAD In Mammalian Cells
A further complication regarding the assessment of the intracellular NAD concentration is brought about by the subcellular compartmentation. It has been estimated that up to 70% of the total cellular NAD can be sequestered within mitochondria. Apparently, the relative amount of mitochondria within a tissue correlates with the share of mitochondrial (versus cytosolic) NAD. As a result, the mitochondrial fraction of NAD is very high in heart and other mitochondria-rich tissues (brain, liver). The other cellular pool of NAD is considered to be cytosolic, freely exchangeable with the nucleus. Since there appears to be no physiological pathway for the exchange between cytosolic and mitochondrial NAD (except in plants), these two pools have to be regarded as independent of one another. However, the redox ratio is transmitted between these two compartments by means of transport systems for metabolites that are substrates of NAD-dependent dehydrogenases present in both compartments. For example, the well known malate-aspartate shuttle system carries redox equivalents (but not NADH itself) from the cytosol into mitochondria.

Compartmentation of NAD Metabolism
Given two major independent NAD pools, cytosolic and mitochondrial, it would be reasonable to expect a corresponding localisation of the biosynthetic enzymes. Surprisingly, some of the key enzymes of NAD biosynthesis are localised within the nucleus. In yeast, both nicotinate phosphoribosyltransferase and an isoform of NMNAT have been localised to the nucleus. The predominant human isoform of NMNAT (hNMNAT1) is also located within the nucleus. It is still puzzling why presumed simple metabolic enzymes are present within the nucleus. However, the discovery of nuclear NAD-dependent signalling events, such as poly-ADP-ribosylation and histone deacetylation by sirtuins, provides at least a rationale for the constant need for NAD in this compartment. Despite the fact that nuclear NAD is supposed to freely permeate into the cytosol, a second human isoform of NMNAT exists and appears to be cytosolic. On the other hand, the pathway of mitochondrial NAD generation is still obscure. NMNAT activity in mitochondria has been reported and a third human NMNAT isoform could possibly be localised within these organelles. However, it is unknown how NMN would enter the mitochondria or be formed therein. Fortunately, there is at least no question as to how the second substrate of NMNAT, namely ATP, is generated within mitochondria.

Figure: Compartmentation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)] biosynthesis and major NAD(P)-mediated signalling pathways in eukaryotic cells. The major reactions of NAD(P)-mediated signalling and potential compartmentation of the final steps of NAD(P) biosynthesis are summarized. Not all pathways occur necessarily in the same cell. The question mark indicates that the subcellular localization of the enzyme has not been clearly established. Abbreviations: ART, mono-ADP-ribosyltransferase; TCA, citric acid cycle; cADPR, cyclic ADP-ribose; ER, endoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate; NADase, bifunctional NAD glycohydrolase/ADP-ribosyl cyclase; NMNAT, nicotinamide mononucleotide adenylyltransferase; PARP, poly-ADP-ribose polymerase. .

References:

Reviews:

M. Ziegler (2000): New functions of a long known molecule - Emerging roles of NAD in cellular signaling. Eur. J. Biochem. 267, 1550-1564

M. Ziegler, S. L. Oei (2001): A cellular survival switch: Poly(ADP-ribosyl)ation stimulates DNA repair and silences transcription. BioEssays 23, 543-548

F. Di Lisa, M. Ziegler (2001): Pathophysiological relevance of mitochondrial NAD metabolism. FEBS Lett. 492, 4-8

F. Berger, M.-H. Ramirez-Hernandez, M.Ziegler (2004): The new life of a centenarian: signalling functions of NAD(P). Trends Biochem. Sci. 29, 111-118

L. Hunt, F. Lerner, M. Ziegler (2004): NAD ö new roles in signalling and gene regulation in plants. New Phytologist 163, 31-44

M. Niere, M. Ziegler (2004): NAD surfaces again. Biochem. J. 382, e4-e5

M. Ziegler (2004): NAD - metabolism and regulatory functions. in: Poly(ADP-ribosyl)ation (BŸrkle ed.), Landes Bioscience

Selected original articles: Ê

M. Ziegler, D. Jorcke, M. Schweiger (1997): Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase. Biochem. J. 326, 401-405

S. L. Oei, J. Griesenbeck, M. Ziegler, M. Schweiger (1998): A novel function of poly(ADP-ribosyl)ation: Silencing of RNA polymerase II-dependent transcription. Biochemistry 37, 1465-1469

S. L. Oei, J. Griesenbeck, M. Schweiger, M. Ziegler (1998): Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors. J. Biol. Chem. 273, 31644-31647

S. L. Oei, M. Ziegler (2000): ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose). J. Biol. Chem. 275, 23234ö23239

M. Schweiger, K. Hennig, M. Niere, F. Lerner, C. Weise, T. Specht, M. Hirsch-Kauffmann, S. L. Oei, M. Ziegler (2001): Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase, an essential enzyme in NAD synthesis. FEBS Lett. 492, 95-100

A. Herrero-Yraola, S. Bakhit, P. Franke, C. Weise, M. Schweiger, D. Jorcke, M. Ziegler (2001): Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria. EMBO J. 20, 2404-2412

F. Lerner, M. Niere, A. Ludwig, M. Ziegler (2001): Structural and functional characterization of human NAD kinase. Biochem. Biophys. Res. Communic. 288, 69-74

E. Werner, M. Ziegler, F. Lerner, M.Schweiger, U. Heinemann (2002): Crystal structure of human nicotinamide mononucleotide adenylyltransferase in complex with NMN. FEBS Lett., 516, 239-244

E. Petermann, M. Ziegler, S. L. Oei (2003): ATP-dependent selection between long-patch and single-nucleotide base excision repair. DNA Repair 2, 1101-1114