An anchor-tether 'hindered' HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model.

BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Triple-Isotope Tracing for Pathway Discernment of NMN-Induced NAD+ Biosynthesis in Whole Mice.

Numerous efforts in basic and clinical studies have explored the potential anti-aging and health-promoting effects of NAD+-boosting compounds such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). Despite these extensive efforts, our understanding and characterization of their whole-body pharmacodynamics, impact on NAD+ tissue distribution, and mechanism of action in various tissues remain incomplete. In this study, we administered NMN via intraperitoneal injection or oral gavage and conducted a rigorous evaluation of NMN's pharmacodynamic effects on whole-body NAD+ homeostasis in mice. To provide more confident insights into NMN metabolism and NAD+ biosynthesis across different tissues and organs, we employed a novel approach using triple-isotopically labeled [18O-phosphoryl-18O-carbonyl-13C-1-ribosyl] NMN. Our results provide a more comprehensive characterization of the NMN impact on NAD+ concentrations and absolute amounts in various tissues and the whole body. We also demonstrate that mice primarily rely on the nicotinamide and NR salvage pathways to generate NAD+ from NMN, while the uptake of intact NMN plays a minimal role. Overall, the tissue-specific pharmacodynamic effects of NMN administration through different routes offer novel insights into whole-body NAD+ homeostasis, laying a crucial foundation for the development of NMN as a therapeutic supplement in humans.

Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and in vivo.

Interest in pharmacological agents capable of increasing cellular NAD+ concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD+ concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD+ concentrations by 2.5-10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD+ increases at equivalent concentrations. NRH also provides substantial NAD+ increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD+/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD+-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD+ consumption, suggesting that it acts as a biochemical precursor to NAD+ Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD+ and a potent pharmacologic agent for NAD+ concentration enhancement in cells and tissues.

Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

NAD(+) metabolism: Bioenergetics, signaling and manipulation for therapy.

We survey the historical development of scientific knowledge surrounding Vitamin B3, and describe the active metabolite forms of Vitamin B3, the pyridine dinucleotides NAD+ and NADP+ which are essential to cellular processes of energy metabolism, cell protection and biosynthesis. The study of NAD+ has become reinvigorated by new understandings that dynamics within NAD+ metabolism trigger major signaling processes coupled to effectors (sirtuins, PARPs, and CD38) that reprogram cellular metabolism using NAD+ as an effector substrate. Cellular adaptations include stimulation of mitochondrial biogenesis, a process fundamental to adjusting cellular and tissue physiology to reduced nutrient availability and/or increased energy demand. Several mammalian metabolic pathways converge to NAD+, including tryptophan-derived de novo pathways, nicotinamide salvage pathways, nicotinic acid salvage and nucleoside salvage pathways incorporating nicotinamide riboside and nicotinic acid riboside. Key discoveries highlight a therapeutic potential for targeting NAD+ biosynthetic pathways for treatment of human diseases. A recent emergence of understanding that NAD+ homeostasis is vulnerable to aging and disease processes has stimulated testing to determine if replenishment or augmentation of cellular or tissue NAD+ can have ameliorative effects on aging or disease phenotypes. This experimental approach has provided several proofs of concept successes demonstrating that replenishment or augmentation of NAD+ concentrations can provide ameliorative or curative benefits. Thus NAD+ metabolic pathways can provide key biomarkers and parameters for assessing and modulating organism health.

Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.

UNLABELLED: With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD(+) ) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic ß-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 knockout mice (Sirt1(hep-/-) ), whereas apolipoprotein E-deficient mice (Apoe(-/-) ) challenged with a high-fat high-cholesterol diet affirmed the use of NR in other independent models of NAFLD. CONCLUSION: Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD.

Multifunctionalization of cetuximab with bioorthogonal chemistries and parallel EGFR profiling of cell-lines using imaging, FACS and immunoprecipitation approaches.

The ability to derivatize antibodies is currently limited by the chemical structure of antibodies as polypeptides. Modern methods of bioorthogonal and biocompatible chemical modifications could make antibody functionalization more predictable and easier, without compromising the functions of the antibody. To explore this concept, we modified the well-known anti-epidermal growth factor receptor (EGFR) drug, cetuximab (Erbitux®), with 5-azido-2-nitro-benzoyl (ANB) modifications by optimization of an acylation protocol. We then show that the resulting ANB-cetuximab can be reliably modified with dyes (TAMRA and carboxyrhodamine) or a novel synthesized cyclooctyne modified biotin. The resulting dye- and biotin-modified cetuximabs were then tested across several assay platforms with several cell lines including U87, LN229, F98EGFR, F98WT and HEK293 cells. The assay platforms included fluorescence microscopy, FACS and biotin-avidin based immunoprecipitation methods. The modified antibody performs consistently in all of these assay platforms, reliably determining relative abundances of EGFR expression on EGFR expressing cells (LN229 and F98EGFR) and failing to cross react with weak to negative EGFR expressing cells (U87, F98WT and HEK293). The ease of achieving diverse and assay relevant functionalizations as well as the consequent rapid construction of highly correlated antigen expression data sets highlights the power of bioorthogonal and biocompatible methods to conjugate macromolecules. These data provide a proof of concept for a multifunctionalization strategy that leverages the biochemical versatility and antigen specificity of antibodies.

Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection.

PURPOSE OF REVIEW: This review focuses upon the biology and metabolism of a trace component in foods called nicotinamide riboside. Nicotinamide riboside is a precursor of nicotinamide adenine dinucleotide (NAD), and is a source of Vitamin B3. Evidence indicates that nicotinamide riboside has unique properties as a Vitamin B3. We review knowledge of the metabolism of this substance, as well as recent work suggesting novel health benefits that might be associated with nicotinamide riboside taken in larger quantities than is found naturally in foods. RECENT FINDINGS: Recent work investigating the effects of nicotinamide riboside in yeast and mammals established that it is metabolized by at least two types of metabolic pathways. The first of these is degradative and produces nicotinamide. The second pathway involves kinases called nicotinamide riboside kinases (Nrk1 and Nrk2, in humans). The likely involvement of the kinase pathway is implicated in the unique effects of nicotinamide riboside in raising tissue NAD concentrations in rodents and for potent effects in eliciting insulin sensitivity, mitochondrial biogenesis, and enhancement of sirtuin functions. Additional studies with nicotinamide riboside in models of Alzheimer's disease indicate bioavailability to brain and protective effects, likely by stimulation of brain NAD synthesis. SUMMARY: Initial studies have clarified the potential for a lesser-known Vitamin B3 called nicotinamide riboside that is available in selected foods, and possibly available to humans by supplements. It has properties that are insulin sensitizing, enhancing to exercise, resisting to negative effects of high-fat diet, and neuroprotecting.

Metabolic Disease, NAD Metabolism, Nicotinamide Riboside, and the Gut Microbiome: Connecting the Dots from the Gut to Physiology.

The effort to use nutrients as interventions to treat human disease has been important to medicine. A current example in this vein pertains to NAD+ boosters, such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), which are in many clinical trials in a variety of disease conditions. Independent laboratories have shown that ingested NR (or NMN) has mitigating effects on metabolic syndrome in mice. V. V. Lozada-Fernández, O. deLeon, S. L. Kellogg, F. L. Saravia, et al. (mSystems 7:e00230-21, 2022, https://doi.org/10.1128/mSystems.00230-21) show that NR shifts gut microbiome contents and that the transplantation of an NR-conditioned microbiome by fecal transfer reproduces some effects of NR in mice on a high-fat diet. The involvement of the gut microbiome as a factor in NR effects is linked to changes to the gut microbiome and its activity to transform NR and downstream catabolites. This commentary draws attention to these findings and focuses on some puzzling aspects of NAD+ boosters, exploring the still murky interactions between NAD+ metabolism, energy homeostasis, and the gut microbiome.

Identification of the aryl hydrocarbon receptor target gene TiPARP as a mediator of suppression of hepatic gluconeogenesis by 2,3,7,8-tetrachlorodibenzo-p-dioxin and of nicotinamide as a corrective agent for this effect.

The environmental toxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin) produces diverse toxic effects including a lethal wasting syndrome whose hallmark is suppressed hepatic gluconeogenesis. All TCDD toxicities require activation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Whereas the mechanism for AHR induction of target genes is well understood, it is not known how AHR activation produces any TCDD toxicity. This report identifies for the first time an AHR target gene, TiPARP (TCDD-inducible poly(ADP-ribose) polymerase, PARP7) that can mediate a TCDD toxicity, i.e. suppression of hepatic gluconeogenesis. TCDD suppressed hepatic glucose production, expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), and NAD(+) levels, and increased PARP activity and TiPARP expression. TCDD also increased acetylation and ubiquitin-dependent proteosomal degradation of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), a coactivator of PEPCK and G6Pase transcription. TiPARP overexpression reproduced TCDD effects on glucose output and NAD(+) levels whereas TiPARP silencing diminished them. TiPARP overexpression also increased PGC1α acetylation and decreased PGC1α levels. In contrast, silencing of cytochromes P450 (CYP) 1A, main AHR-induced genes, did not alter TCDD suppression of gluconeogenesis. The vitamin B3 constituent, nicotinamide (NAM), prevented TCDD suppression of glucose output, NAD(+), and gluconeogenic genes and stabilized PGC1α. The corrective effects of NAM could be attributed to increased NAD(+) levels and suppression of AHR target gene induction. The results reveal that TiPARP can mediate a TCDD effect, that the AHR is linked to PGC1α function and stability and that NAM has novel AHR antagonist activity.

Sirtuin chemical mechanisms.

Sirtuins are ancient proteins widely distributed in all lifeforms of earth. These proteins are universally able to bind NAD(+), and activate it to effect ADP-ribosylation of cellular nucleophiles. The most commonly observed sirtuin reaction is the ADP-ribosylation of acetyllysine, which leads to NAD(+)-dependent deacetylation. Other types of ADP-ribosylation have also been observed, including protein ADP-ribosylation, NAD(+) solvolysis and ADP-ribosyltransfer to 5,6-dimethylbenzimidazole, a reaction involved in eubacterial cobalamin biosynthesis. This review broadly surveys the chemistries and chemical mechanisms of these enzymes.

Mechanism-based affinity capture of sirtuins.

The ability to probe for catalytic activities of enzymes and to detect their abundance in complex biochemical contexts has traditionally relied on a combination of kinetic assays and techniques such as western blots that use expensive reagents such as antibodies. The ability to simultaneously detect activity and isolate a protein catalyst from a mixture is even more difficult and currently impossible in most cases. In this manuscript we describe a chemical approach that achieves this goal for a unique family of enzymes called sirtuins using novel chemical tools, enabling rapid detection of activity and isolation of these protein catalysts. Sirtuin deacetylases are implicated in the regulation of many physiological functions including energy metabolism, DNA-damage response, and cellular stress resistance. We synthesized an aminooxy-derivatized NAD(+) and a pan-sirtuin inhibitor that reacts on sirtuin active sites to form a chemically stable complex that can subsequently be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight determination. We show that these tools are cross reactive against a variety of human sirtuin isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can react with microbial derived sirtuins as well. Finally, we demonstrate the ability to simultaneously detect multiple sirtuin isoforms in reaction mixtures with this methodology, establishing proof of concept tools for chemical studies of sirtuins in complex biological samples.

Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.

NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.

High-resolution crystal structures of Streptococcus pneumoniae nicotinamidase with trapped intermediates provide insights into the catalytic mechanism and inhibition by aldehydes .

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD(+) in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD(+) homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD(+)-consuming enzymes, such as the NAD(+)-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.

Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 µM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid (18)O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.

Global analysis of transcriptional regulation by poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in MCF-7 human breast cancer cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.

Transition state of ADP-ribosylation of acetyllysine catalyzed by Archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches.

Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes bind NAD(+), a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD(+)-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD(+) with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1(N)-(15)N] = 1.024(2), [1'(N)-(14)C] = 1.014(4), [1'(N)-(3)H] = 1.300(3), [2'(N)-(3)H] = 1.099(5), [4'(N)-(3)H] = 0.997(2), [5'(N)-(3)H] = 1.020(5), [4'(N)-(18)O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase.

Dihydronicotinamide riboside (NRH) has been suggested to act as a precursor for the synthesis of NAD+, but the biochemical pathway converting it has been unknown. Here, we show that NRH can be converted into NAD+ via a salvage pathway in which adenosine kinase (ADK, also known as AK) acts as an NRH kinase. Using isotope-labelling approaches, we demonstrate that NRH is fully incorporated into NAD+, with NMNH acting as an intermediate. We further show that AK is enriched in fractions from cell lysates with NRH kinase activity, and that AK can convert NRH into NAD+. In cultured cells and mouse liver, pharmacological or genetic inhibition of AK blocks formation of reduced nicotinamide mononucleotide (NMNH) and inhibits NRH-stimulated NAD+ biosynthesis. Finally, we confirm the presence of endogenous NRH in the liver with metabolomics. Our findings establish NRH as a natural precursor of NAD+ and reveal a new route for NAD+ biosynthesis via an NRH salvage pathway involving AK.