RESUMO
Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.
Assuntos
Alcaloides , Sarcopenia , Humanos , Masculino , Camundongos , Animais , Sarcopenia/tratamento farmacológico , Sarcopenia/prevenção & controle , Sarcopenia/metabolismo , NAD/metabolismo , Caenorhabditis elegans , Envelhecimento , Músculo Esquelético/metabolismo , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Alcaloides/metabolismoRESUMO
We report the synthesis of vitamin B1, B2, and B3 derived nucleotides and dinucleotides generated either through mechanochemical or solution phase chemistry. Under the explored conditions, adenosine and thiamine proved to be particularly amenable to milling conditions. Following optimization of the chemistry related to the formation pyrophosphate bonds, mixed dinucleotides of adenine and thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide riboside and 3-carboxamide 4-pyridone riboside (both vitamin B3 derivatives) were generated in good yields. Furthermore, we report an efficient synthesis of the MW+4 isotopologue of NAD+ for which deuterium incorporation is present on either side of the dinucleotidic linkage, poised for isotopic tracing experiments by mass spectrometry. Many of these mixed species are novel and present unexplored possibilities to simultaneously enhance or modulate cofactor transporters and enzymes of independent biosynthetic pathways.
Assuntos
Niacina , Niacina/metabolismo , Riboflavina , Tiamina/análiseRESUMO
Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.
RESUMO
Assembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase ß (Polß) and XRCC1 to DNA lesion sites identifies a role for Polß in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polß's dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polß and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polß abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.
Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA de Neoplasias/metabolismo , NAD/metabolismo , Neoplasias/enzimologia , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sirtuínas/metabolismo , Células A549 , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA de Neoplasias/genética , Humanos , Cinética , Microscopia Confocal , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Sirtuínas/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismoRESUMO
Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.
Assuntos
Metaboloma , NAD/biossíntese , Plasma/metabolismo , Soro/metabolismo , Espectrometria de Massas em Tandem/métodos , Urina/fisiologia , Animais , Doadores de Sangue , Cromatografia Líquida/métodos , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Monitorização Fisiológica/métodos , Oxirredução , Projetos Piloto , Plasma/química , Soro/química , Urina/químicaRESUMO
Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , NAD+ Nucleosidase/química , NAD+ Nucleosidase/isolamento & purificação , NAD+ Nucleosidase/metabolismo , ADP-Ribosil Ciclase/metabolismo , Animais , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Cristalografia por Raios X , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , NAD/metabolismo , NAD+ Nucleosidase/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/patogenicidade , Conformação Proteica , Células Sf9 , Transdução de SinaisRESUMO
All life forms require nicotinamide adenine dinucleotide, NAD+, and its reduced form NADH. They are redox partners in hundreds of cellular enzymatic reactions. Changes in the intracellular levels of total NAD (NAD+ + NADH) and the (NAD+/NADH) ratio can cause cellular dysfunction. When not present in protein complexes, NADH and its phosphorylated form NADPH degrade through intricate mechanisms. Replenishment of a declining total NAD pool can be achieved with biosynthetic precursors that include one of the reduced forms of nicotinamide riboside (NR+), NRH. NRH, like NADH and NADPH, is prone to degradation via oxidation, hydration, and isomerization and, as such, is an excellent model compound to rationalize the nonenzymatic metabolism of NAD(P)H in a biological context. Here, we report on the stability of NRH and its propensity to isomerize and irreversibly degrade. We also report the preparation of two of its naturally occurring isomers, their chemical stability, their reactivity toward NRH-processing enzymes, and their cell-specific cytotoxicity. Furthermore, we identify a mechanism by which NRH degradation causes covalent peptide modifications, a process that could expose a novel type of NADH-protein modifications and correlate NADH accumulation with "protein aging." This work highlights the current limitations in detecting NADH's endogenous catabolites and in establishing the capacity for inducing cellular dysfunction.
Assuntos
Niacinamida/análogos & derivados , Compostos de Piridínio/química , Isomerismo , NAD/química , Niacinamida/química , OxirreduçãoRESUMO
Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.
Assuntos
Ácidos Graxos Ômega-3/administração & dosagem , Hipertrigliceridemia/dietoterapia , Triglicerídeos/metabolismo , Amidoidrolases/deficiência , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Bile/metabolismo , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/análogos & derivados , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Humanos , Hipertrigliceridemia/metabolismo , Hipolipemiantes/administração & dosagem , Hipolipemiantes/metabolismo , Absorção Intestinal/efeitos dos fármacos , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação Puntual , Taurina/análogos & derivados , Taurina/metabolismoRESUMO
As catabolites of nicotinamide possess physiological relevance, pyridones are often included in metabolomics measurements and associated with pathological outcomes in acute kidney injury (AKI). Pyridones are oxidation products of nicotinamide, its methylated form, and its ribosylated form. While they are viewed as markers of over-oxidation, they are often wrongly reported or mislabeled. To address this, we provide a comprehensive characterization of these catabolites of vitamin B3, justify their nomenclature, and differentiate between the biochemical pathways that lead to their generation. Furthermore, we identify an enzymatic and a chemical process that accounts for the formation of the ribosylated form of these pyridones, known to be cytotoxic. Finally, we demonstrate that the ribosylated form of one of the pyridones, the 4-pyridone-3-carboxamide riboside (4PYR), causes HepG3 cells to die by autophagy; a process that occurs at concentrations that are comparable to physiological concentrations of this species in the plasma in AKI patients.
Assuntos
NAD/metabolismo , Niacinamida/metabolismo , Piridonas/metabolismo , Autofagia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Piridonas/química , Piridonas/farmacologia , Piridonas/uso terapêuticoRESUMO
Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.
Assuntos
Amidas/metabolismo , Vias Biossintéticas , Mamíferos/microbiologia , Mycoplasma/fisiologia , NAD/metabolismo , Administração Oral , Animais , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Metabolismo Energético , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamidase/metabolismo , Mononucleotídeo de Nicotinamida/administração & dosagem , Mononucleotídeo de Nicotinamida/química , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , Compostos de Piridínio/metabolismoRESUMO
OBJECTIVE: A decay in intracellular NAD+ levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD+ precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD+ precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD+ biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD+ precursors that can overcome these limitations. METHODS: Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD+ precursor in mammalian cells and mice. RESULTS: NRH acts as a more potent and faster NAD+ precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD+, thus revealing a new NRK1-independent pathway for NAD+ synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury. CONCLUSIONS: Our data identify a new pathway for NAD+ synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD+ levels.
Assuntos
NAD/biossíntese , NAD/metabolismo , Niacinamida/análogos & derivados , Animais , Linhagem Celular , Masculino , Camundongos , Niacinamida/metabolismo , Niacinamida/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool) , Compostos de Piridínio , RatosRESUMO
The ß-anomeric form of nicotinamide riboside (NR+) is a precursor for nicotinamide adenine dinucleotide (NAD+), a redox cofactor playing a critical role in cell metabolism. Recently, it has been demonstrated that its chloride salt (NR+Cl-) has beneficial effects, and now NR+Cl- is available as a dietary supplement. Syntheses and studies of analogues and derivatives of NR+ are of high importance to unravel the role of NR+ in biochemical processes in living cells and to elaborate the next generation of NR+ derivatives and conjugates with the view of developing novel drug and food supplement candidates. This review provides an overview of the synthetic approaches, the chemical properties, and the structural and functional modifications which have been undertaken on the nicotinoyl riboside scaffold.
RESUMO
The functional cofactors derived from vitamin B3 are nicotinamide adenine dinucleotide (NAD+), its phosphorylated form, nicotinamide adenine dinucleotide phosphate (NADP+) and their reduced forms (NAD(P)H). These cofactors, together referred as the NAD(P)(H) pool, are intimately implicated in all essential bioenergetics, anabolic and catabolic pathways in all forms of life. This pool also contributes to post-translational protein modifications and second messenger generation. Since NAD+ seats at the cross-road between cell metabolism and cell signaling, manipulation of NAD+ bioavailability through vitamin B3 supplementation has become a valuable nutritional and therapeutic avenue. Yet, much remains unexplored regarding vitamin B3 metabolism. The present review highlights the chemical diversity of the vitamin B3-derived anabolites and catabolites of NAD+ and offers a chemical perspective on the approaches adopted to identify, modulate and measure the contribution of various precursors to the NAD(P)(H) pool.
Assuntos
Metaboloma/fisiologia , Niacinamida/metabolismo , Animais , Humanos , Metaboloma/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
The synthesis and the antitumor activity and fluorescent properties screening of novel bisphosphonate conjugates with cytotoxic 3,5-bis((hetero)arylidene)-4-piperidone residues were performed. The facile and rapid synthetic route was based on the aza-Michael addition of NH-3,5-bis((hetero)arylidene)-4-piperidones to tetraethyl ethylidenebisphosphonate. The synthesized compounds displayed high inhibitory properties towards Caov3, A549, PC3, and KB 3-1 human carcinoma cell lines. Among those, compounds bearing 4-cyano-phenyl and 3-pyridinyl substituents were revealed as the most active drug candidates with IC(50) values in the range of 0.5-2.5 µM. Methylenebisphosphonate with 4-Me(2) N-C(6) H(4) groups in the piperidone framework possessing fluorescence properties may be of interest for visualization of BPs skeletal distribution and cellular uptake in bones and other tissues.