RESUMEN
Nicotinamide adenine dinucleotide, in its oxidized (NAD+) and reduced (NADH) forms, is a reduction-oxidation (redox) co-factor and substrate for signalling enzymes that have essential roles in metabolism. The recognition that NAD+ levels fall in response to stress and can be readily replenished through supplementation has fostered great interest in the potential benefits of increasing or restoring NAD+ levels in humans to prevent or delay diseases and degenerative processes. However, much about the biology of NAD+ and related molecules remains poorly understood. In this Review, we discuss the current knowledge of NAD+ metabolism, including limitations of, assumptions about and unappreciated factors that might influence the success or contribute to risks of NAD+ supplementation. We highlight several ongoing controversies in the field, and discuss the role of the microbiome in modulating the availability of NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), the presence of multiple cellular compartments that have distinct pools of NAD+ and NADH, and non-canonical NAD+ and NADH degradation pathways. We conclude that a substantial investment in understanding the fundamental biology of NAD+, its detection and its metabolites in specific cells and cellular compartments is needed to support current translational efforts to safely boost NAD+ levels in humans.
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Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it's estimated that up to 75â¯% of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD+-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polß), a bifunctional DNA polymerase containing both DNA synthesis and 5'-deoxyribose-phosphate lyase (5'dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD+ metabolism, but still produce sufficient nuclear NAD+ for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polß protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.
Asunto(s)
ADN Polimerasa beta , Glutaratos , Isocitrato Deshidrogenasa , ADN Polimerasa beta/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/genética , Glutaratos/metabolismo , Línea Celular Tumoral , Reparación del ADN , Antineoplásicos Alquilantes/farmacología , Temozolomida/farmacología , Mutación , Glioma/metabolismo , Glioma/genética , Glioma/tratamiento farmacológico , Alquilantes/farmacología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Daño del ADNRESUMEN
Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.
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Autofagia , Células Madre Pluripotentes Inducidas , NAD , Enfermedad de Niemann-Pick Tipo C , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Humanos , Autofagia/efectos de los fármacos , NAD/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Memantina/farmacología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Mitofagia/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
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.
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Alcaloides , Sarcopenia , Humanos , Masculino , Ratones , Animales , Sarcopenia/tratamiento farmacológico , Sarcopenia/prevención & control , Sarcopenia/metabolismo , NAD/metabolismo , Caenorhabditis elegans , Envejecimiento , Músculo Esquelético/metabolismo , Alcaloides/farmacología , Alcaloides/uso terapéutico , Alcaloides/metabolismoRESUMEN
Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD+) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD+, the key redox cofactor in metabolism and bioenergetics maintenance.
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NAD , Nucleótidos , Humanos , NAD/metabolismo , Nucleótidos/metabolismo , Nucleósidos/metabolismo , Metabolismo Energético , PiridonasRESUMEN
Dietary vitamin B3 components, such as nicotinamide and nicotinic acid, are precursors to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+). NAD+ levels are thought to decline with age and disease. While the drivers of this decline remain under intense investigation, strategies have emerged seeking to functionally maintain NAD+ levels through supplementation with NAD+ biosynthetic intermediates. These include marketed products, such as nicotinamide riboside (NR) and its phosphorylated form (NMN). More recent developments have shown that NRH (the reduced form of NR) and its phosphorylated form NMNH also increases NAD+ levels upon administration, although they initially generate NADH (the reduced form of NAD+). Other means to increase the combined levels of NAD+ and NADH, NAD(H), include the inhibition of NAD+-consuming enzymes or activation of biosynthetic pathways. Multiple studies have shown that supplementation with an NAD(H) precursor changes the profile of NAD(H) catabolism. Yet, the pharmacological significance of NAD(H) catabolites is rarely considered although the distribution and abundance of these catabolites differ depending on the NAD(H) precursor used, the species in which the study is conducted, and the tissues used for the quantification. Significantly, some of these metabolites have emerged as biomarkers in physiological disorders and might not be innocuous. Herein, we review the known and emerging catabolites of the NAD(H) metabolome and highlight their biochemical and physiological function as well as key chemical and biochemical reactions leading to their formation. Furthermore, we emphasize the need for analytical methods that inform on the full NAD(H) metabolome since the relative abundance of NAD(H) catabolites informs how NAD(H) precursors are used, recycled, and eliminated.
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NAD , Niacina , NAD/metabolismo , Niacinamida/metabolismo , Metaboloma , Oxidación-Reducción , Biomarcadores/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.
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NAD , Niacina , Ratones , Animales , Niacinamida/farmacología , MamíferosRESUMEN
Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning.
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Dihidroxiacetona , Sistemas Electrónicos de Liberación de Nicotina , Dihidroxiacetona/farmacología , Especies Reactivas de Oxígeno , Mitocondrias , HígadoRESUMEN
Pyridone adenine dinucleotides (ox-NADs) are redox inactive derivatives of the enzyme cofactor and substrate nicotinamide adenine dinucleotide (NAD) that have a carbonyl group at the C2, C4, or C6 positions of the nicotinamide ring. These aberrant cofactor analogs accumulate in cells under stress and are potential inhibitors of enzymes that use NAD(H). We studied the conformational landscape of ox-NADs in solution using molecular dynamics simulations. Compared to NAD+ and NADH, 2-ox-NAD and 4-ox-NAD have an enhanced propensity for adopting the anti conformation of the pyridone ribose group, whereas 6-ox-NAD exhibits greater syn potential. Consequently, 2-ox-NAD and 4-ox-NAD have increased preference for folding into compact conformations, whereas 6-ox-NAD is more extended. ox-NADs have distinctive preferences for the orientation of the pyridone amide group, which are driven by intramolecular hydrogen bonding and steric interactions. These conformational preferences are compared to those of protein-bound NAD(H). Our results may help in identifying enzymes targeted by ox-NADs.
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Simulación de Dinámica Molecular , NAD , Adenina , Amidas , Dapsona/análogos & derivados , NAD/metabolismo , Niacinamida , Piridonas , RibosaRESUMEN
Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.
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NAD , Purina-Nucleósido Fosforilasa , Humanos , Ratones , Animales , NAD/metabolismo , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Niacinamida/farmacología , Niacinamida/metabolismo , Compuestos de Piridinio , Mamíferos/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway. These resistance mechanisms are either somatic or arise in response to treatment, highlighting the need to uncover treatments to overcome resistance. We found that administration of the NAD+ precursor dihydronicotinamide riboside (NRH) to raise cellular NAD+ levels combined with PARG inhibition (PARGi) triggers hyperaccumulation of poly(ADP-ribose) (PAR), resulting from both DNA damage-induced and replication-stress-induced PARP1 activation. Here, we show that the NRH/PARGi combination enhances the cytotoxicity of TMZ. Specifically, NRH rapidly increases NAD+ levels in both TMZ-sensitive and TMZ-resistant GBM-derived cells and enhances the accumulation of PAR following TMZ treatment. Furthermore, NRH promotes hyperaccumulation of PAR in the presence of TMZ and PARGi. This combination strongly suppresses the cell growth of GBM cells depleted of MSH6 or cells expressing MGMT, suggesting that this regimen may improve the efficacy of TMZ to overcome treatment resistance in GBM.
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Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD+), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD+ precursors influence intracellular NAD+ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD+ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD+ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD+ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD+ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD+ precursor in mammalian cells and how different NAD+ precursors can interact and influence each other when co-administered.
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NAD , Niacinamida , Animales , Mamíferos , Ratones , Niacinamida/análogos & derivados , Compuestos de PiridinioRESUMEN
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.
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Niacina , Niacina/metabolismo , Riboflavina , Tiamina/análisisRESUMEN
This article contains a synthetic protocol for solvent-assisted mechanochemical synthesis of a nucleotide dimer. First, a dinucleoside phosphite is prepared by solvent-assisted mechanochemistry via the phosphoramidite method. Second, the dinucleoside phosphite is oxidized to form the dinucleotide under mechanochemical conditions. Finally, the dinucleotide is purified by column chromatography. Support protocols are also provided for preparing the acidic salts that can be utilized for phosphoramidite couplings and for demonstrating that the reaction occurs under mechanochemical conditions rather than as a result of solvent added for analysis. Mechanochemistry as applied to synthesis of dinucleotides is a recent development and it is anticipated that the principles in this protocol will be widely applicable to a range of nucleoside and ribonucleoside monomers. The advantages of mechanochemistry over traditional solution-phase chemistry are the simplicity of the procedure, improved hydrolytic stability, and elimination of the need to solubilize poorly soluble compounds. © 2022 Wiley Periodicals LLC. Basic Protocol: Solvent-assisted mechanochemical synthesis of a nucleotide dimer Supplementary Protocol 1: Synthesis of N-methylimidazolium triflate Supplementary Protocol 2: Synthesis of pyridinium trifluoroacetate Supplementary Protocol 3: Confirmation of the efficacy of mechanochemical conditions.
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Nucleótidos , Fosfitos , Nucleósidos , Oligonucleótidos , Polímeros/química , SolventesRESUMEN
Dihydroxyacetone (DHA) is a major byproduct of e-cigarette combustion and is the active ingredient in sunless tanning products. Mounting evidence points to its damaging effects on cellular functions. While developing a simple synthetic route to monomeric [13C3]DHA for flux metabolic studies that compared DHA and glyceraldehyde (GA) metabolism, we uncovered that solid DHA ages upon storage and differences in the relative abundance of each of its isomer occur when reconstituted in an aqueous solution. While all three of the dimeric forms of DHA ultimately resolve to the ketone and hydrated forms of monomeric DHA once in water at room temperature, these species require hours rather than minutes to reach an equilibrium favoring the monomeric species. Consequently, when used in bolus or flux experiments, the relative abundance of each isomer and its effects at the time of application is dependent on the initial DHA isomeric composition and concentration, and time of equilibration in solution before use. Here, we make recommendations for the more consistent handling of DHA as we report conditions that ensure that DHA is present in its monomeric form while in solutions, conditions used in an isotopic tracing study that specifically compared monomeric DHA and GA metabolism in cells.
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Dihidroxiacetona , Sistemas Electrónicos de Liberación de Nicotina , Isomerismo , SolucionesRESUMEN
Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD+ levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD+ precursors can promote inflammation in macrophages. The effect of NRH in NAD+ boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IκB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment.
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NAD , Niacinamida , Citocinas , Glicósidos , Macrófagos/metabolismo , NAD/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacología , FenotipoRESUMEN
Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.
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Aldehído Oxidorreductasas/antagonistas & inhibidores , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Tretinoina/metabolismo , HumanosRESUMEN
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.
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Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.
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ADP-Ribosil Ciclasa/metabolismo , Antígenos CD/metabolismo , Glicósido Hidrolasas/metabolismo , Niacinamida/análogos & derivados , Compuestos de Piridinio/farmacocinética , Células A549 , ADP-Ribosil Ciclasa/genética , Administración Oral , Envejecimiento/efectos de los fármacos , Animales , Antígenos CD/genética , Suplementos Dietéticos , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Microbioma Gastrointestinal , Glicósido Hidrolasas/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestino Delgado/metabolismo , Intestino Delgado/microbiología , Ratones , Ratones Noqueados , Niacina/metabolismo , Niacinamida/administración & dosificación , Niacinamida/metabolismo , Niacinamida/farmacocinética , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Compuestos de Piridinio/administración & dosificaciónRESUMEN
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.