Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K _m and V _max values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min⁻¹ mg⁻¹ protein, respectively. The enzyme’s pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.     

Inhibition of Nicotinamide Phosphoribosyltransferase: CELLULAR BIOENERGETICS REVEALS A MITOCHONDRIAL INSENSITIVE NAD POOL*

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.     

Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix

Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific “knock-down” of the NAD content which is readily detectable by PAR formation.     

The Regulatory Role of NAD in Human and Animal Cells

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD⁺ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD⁺ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.     

Phosphate-responsive signaling pathway is a novel component of NAD+ metabolism in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.     

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.     

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.     

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.     

A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.     

Isoform-specific Targeting and Interaction Domains in Human Nicotinamide Mononucleotide Adenylyltransferases

Several important signaling pathways require NAD as substrate, thereby leading to significant consumption of the molecule. Because NAD is also an essential redox carrier, its continuous resynthesis is vital. In higher eukaryotes, maintenance of compartmentalized NAD pools is critical, but so far rather little is known about the regulation and subcellular distribution of NAD biosynthetic enzymes. The key step in NAD biosynthesis is the formation of the dinucleotide by nicotinamide/nicotinic acid mononucleotide adenylyltransferases (NMNATs). The three human isoforms were localized to the nucleus, the Golgi complex, and mitochondria. Here, we show that their genes contain unique exons that encode isoform-specific domains to mediate subcellular targeting and post-translational modifications. These domains are dispensable for catalytic activity, consistent with their absence from NMNATs of lower organisms. We further demonstrate that the Golgi-associated NMNAT is palmitoylated at two adjacent cysteine residues of its isoform-specific domain and thereby anchored at the cytoplasmic surface, a potential mechanism to regulate the cytosolic NAD pool. Insertion of unique domains thus provides a yet unrecognized enzyme targeting mode, which has also been adapted to modulate subcellular NAD supply.     

Three-minute high-performance liquid chromatographic assay for NMN adenylyltransferase using a 20-mm-long reversed-phase column

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C₁₈ reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22°C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells.     

Synthesis and biological evaluation of NAD analogs as human pyridine nucleotide adenylyltransferase inhibitors

NAD analogs modified at the ribose adenylyl moiety, named N-2'-MeAD and Na-2'-MeAD, were synthesized as ligands of pyridine nucleotide (NMN/NaMN) adenylyltransferase (NMNAT). Both dinucleotides resulted selective inhibitors against human NMNAT-3 isoenzyme.     

Metabolism of NAD+ in the nuclei of human placenta

It has long been known that the major function of NAD+ is as an electron carrier in various biological oxidation-reduction systems. From many papers it is evident that NAD+ is involved as substrate in ADP-ribosylation reactions. We have focused our attention on two chromatin enzymes: NMN-adenylyltransferase that catalyzes reversible synthesis of NAD+ utilizing ATP and NMN, and poly(ADP-ribose)polymerase that covalently modifies nucleosomal proteins through poly ADP-ribosylation reactions. Here we provided evidence of these activities in a system of isolated nuclei from human placenta. The data presented in this report show that purified nuclei might be useful to study the nuclear location of these enzymes and their reciprocal interactions.     

Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms

Nicotinamide mononucleotide adenylyltransferase (NMNAT) is the central enzyme of the NAD biosynthetic pathway. Three human NMNAT isoforms have recently been identified, but isoform-specific functions are presently unknown, although a tissue-specific role has been suggested. Analyses of the subcellular localization confirmed NMNAT1 to be a nuclear protein, whereas NMNAT2 and -3 were localized to the Golgi complex and the mitochondria, respectively. This differential subcellular localization points to an organelle-specific, nonredundant function of each of the three proteins. Comparison of the kinetic properties showed that particularly NMNAT3 exhibits a high tolerance toward substrate modifications. Moreover, as opposed to preferred NAD+ synthesis by NMNAT1, the other two isoforms could also form NADH directly from the reduced nicotinamide mononucleotide, supporting a hitherto unknown pathway of NAD generation. A variety of physiological intermediates was tested and exerted only minor influence on the catalytic activities of the NMNATs. However, gallotannin was found to be a potent inhibitor, thereby compromising its use as a specific inhibitor of poly-ADP-ribose glycohydrolase. The presence of substrate-specific and independent nuclear, mitochondrial, and Golgi-specific NAD biosynthetic pathways is opposed to the assumption of a general cellular NAD pool. Their existence appears to be consistent with important compartment-specific functions rather than to reflect simple functional redundance.     

SYNTHESIS AND BIOLOGICAL EVALUATION OF NAD ANALOGS AS HUMAN PYRIDINE NUCLEOTIDE ADENYLYLTRANSFERASE INHIBITORS

NAD analogs modified at the ribose adenylyl moiety, named N-2′-MeAD and Na-2′-MeAD, were synthesized as ligands of pyridine nucleotide (NMN/NaMN) adenylyltransferase (NMNAT). Both dinucleotides resulted selective inhibitors against human NMNAT-3 isoenzyme.     

Structural characterization of a human cytosolic NMN/NaMN adenylyltransferase and implication in human NAD biosynthesis

Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca(2+) signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.