Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite''s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD⁺). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD⁺ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD⁺ metabolism in these parasites.     

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD⁺ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD⁺ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD⁺ content, we have expressed plant and yeast mitochondrial NAD⁺ carriers in human cells and observed a profound increase in mitochondrial NAD⁺. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD⁺ content. Surprisingly, constitutive redistribution of NAD⁺ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD⁺ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD⁺ levels. These results suggest that a mitochondrial NAD⁺ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD⁺ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.     

Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii

The essential coenzyme nicotinamide adenine dinucleotide (NAD⁺) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD⁺. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD⁺ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD⁺ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD⁺. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD⁺ metabolism and cell longevity.     

The chemical biology of NAD+ regulation in axon degeneration

During axon degeneration, NAD⁺ levels are largely controlled by two enzymes: nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and toll interleukin motif containing protein 1 (SARM1). NMNAT2, which catalyzes the formation of NAD⁺ from NMN and ATP, is actively degraded leading to decreased NAD⁺ levels. SARM1 activity further decreases the concentration of NAD⁺ by catalyzing its hydrolysis to form nicotinamide and a mixture of ADPR and cADPR. Notably, SARM1 knockout mice show decreased neurodegeneration in animal models of axon degeneration, highlighting the therapeutic potential of targeting this novel NAD⁺ hydrolase. This review discusses recent advances in the SARM1 field, including SARM1 structure, regulation, and catalysis as well as the identification of the first SARM1 inhibitors.     

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD⁺ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD⁺ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.     

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD⁺ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD⁺ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD⁺ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD⁺ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD⁺ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD⁺ metabolism in higher eukaryotes.     

NMNAT2‐mediated NAD+ generation is essential for quality control of aged oocytes

Advanced maternal age has been reported to impair oocyte quality; however, the underlying mechanisms remain to be explored. In the present study, we identified the lowered NAD⁺ content and decreased expression of NMNAT2 protein in oocytes from old mice. Specific depletion of NMNAT2 in mouse oocytes disturbs the meiotic apparatus assembly and metabolic activity. Of note, nicotinic acid supplementation during in vitro culture or forced expression of NMNAT2 in aged oocytes was capable of reducing the reactive oxygen species (ROS) production and incidence of spindle/chromosome defects. Moreover, we revealed that activation or overexpression of SIRT1 not only partly prevents the deficient phenotypes of aged oocytes but also ameliorates the meiotic anomalies and oxidative stress in NMNAT2‐depleted oocytes. To sum up, our data indicate a role for NMNAT2 in controlling redox homeostasis during oocyte maturation and uncover that NMNAT2‐ NAD⁺‐SIRT1 is an important pathway mediating the effects of maternal age on oocyte developmental competence.     

NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD⁺ level. NAMPT is a rate-limiting enzyme for NAD⁺ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD⁺ production to maintain mitochondrial NAD⁺ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD⁺ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD⁺ content to promote cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression rescued the mitochondrial membrane potential and content, suggesting that maintained mitochondrial content and integrity by NAMPT overexpression might be one of the key mechanisms to maintain mitochondrial NAD⁺ level and subsequently dictate cell survival under oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and membrane potential.     

Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity

SARM1 is the founding member of the TIR-domain family of NAD⁺ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD⁺ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD⁺ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD⁺ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD⁺ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD⁺ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.     

A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast: THE FIRST ENZYME IN AN INDIRECT AMINOACYLATION PATHWAY*

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA^Gln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA^Glu and tRNA^Gln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA^Gln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.     

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.     

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K⁺ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca²⁺ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca²⁺ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.     

Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD⁺ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD⁺ consuming enzymes. We recently demonstrated that enhancement of NAD⁺/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD⁺ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD⁺ levels promote tumor cell dissemination, we here asked whether inhibition of NAD⁺ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD⁺ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD⁺ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD⁺ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.     

Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD⁺ also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD⁺-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD⁺-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD⁺ by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD⁺ synthesis were overexpressed and led to a 221% increase in the NAD⁺ concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD⁺ content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD⁺ by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD⁺ was constructed, with the intracellular NAD⁺ concentration reaching 26.9 μmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD⁺-producing strain through global regulation metabolic engineering.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD⁺ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD⁺/UDP-GlcA and MoeE5/NAD⁺/UDP-glucose, determined at 1.48 Å and 1.66 Å resolution. The cofactor NAD⁺ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD⁺, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.