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Recent studies highlight the pivotal roles of Nicotinamide adenine dinucleotide (NAD+) and its metabolites in aging and neurodegeneration. Accurate quantification of NAD+ and its metabolite levels in cells or tissues is crucial for advancing biochemical research and interventions targeting aging and neurodegenerative diseases. This study presents an accurate, precise, and rapid LC-MS/MS method using a surrogate matrix to quantify endogenous substances NAD+, nicotinamide mononucleotide (NMN), nicotinamide (NAM), adenosine diphosphate ribose (ADPR), and cyclic adenosine diphosphate ribose (cADPR) concentrations in mice sciatic nerves. Considering the properties of the phosphate groups in the analytes, the column and mobile phase were systematically optimized. These five polar analytes exhibited excellent analytical performance and baseline separation within 5 min on an Atlantis Premier BEH C18 AX column, with methylene phosphonic acid as a mobile phase additive. Enhanced sensitivity addressed the challenges posed by the small sample size of mice sciatic nerve and low NMN and cADPR detection. The method was fully validated, with linear correlation coefficients exceeding 0.992, precision (%relative standard deviation, RSD) values within 8.8%, and accuracy values between 92.2% and 107.3%, suggesting good reproducibility. Analytical recoveries in spiked and diluted matrix ranged from 87.8% to 104.7%, indicating the suitability of water as a surrogate matrix. Application of the method to quantify NAD+ and its metabolite levels in normal and injured mice sciatic nerve identified cADPR as a sensitive biomarker in the nerve injury model. This method is anticipated to deepen our understanding of the connections between NAD+ and its metabolites in health and disease, potentially improving diagnoses of various neurological disorders and aiding drug development for aging and neurodegenerative diseases.
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NAD , Enfermedades Neurodegenerativas , Ratones , Animales , NAD/química , NAD/metabolismo , ADP-Ribosa Cíclica , Cromatografía Liquida , Cromatografía Líquida con Espectrometría de Masas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Nervio Ciático/metabolismoRESUMEN
A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.
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ADP-Ribosil Ciclasa 1 , ADP-Ribosa Cíclica , Animales , Humanos , Ratones , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Células Madre Hematopoyéticas , ADP-Ribosil Ciclasa 1/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismoRESUMEN
Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.
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Proteínas del Dominio Armadillo , Proteínas del Citoesqueleto , Neuronas , Animales , Ratones , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , ADP-Ribosa Cíclica/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , NAD/metabolismo , Neuronas/metabolismoRESUMEN
Adipocytes play a key role in energy storage and homeostasis. Although the role of transcription factors in adipocyte differentiation is known, the effect of endogenous metabolites of low molecular weight remains unclear. Here, we analyzed time-dependent changes in the levels of these metabolites throughout adipocyte differentiation, using metabolome analysis, and demonstrated that there is a positive correlation between cyclic adenosine diphosphate ribose (cADPR) and Pparγ mRNA expression used as a marker of differentiation. We also found that the treatment of C3H10T1/2 adipocytes with cADPR increased the mRNA expression of those marker genes and the accumulation of triglycerides. Furthermore, inhibition of ryanodine receptors (RyR), which are activated by cADPR, caused a significant reduction in mRNA expression levels of the marker genes and triglyceride accumulation in adipocytes. Our findings show that cADPR accelerates adipocytic differentiation via RyR pathway.
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Adipocitos , ADP-Ribosa Cíclica , Ratones , Animales , ADP-Ribosa Cíclica/metabolismo , Adipocitos/metabolismo , Factores de Transcripción/metabolismo , PPAR gamma/metabolismo , Metaboloma , ARN Mensajero/genética , Diferenciación Celular , Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/farmacología , Adipogénesis/genética , Células 3T3-L1RESUMEN
Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced â¼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.
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Asma , Hipersensibilidad Respiratoria , Humanos , Animales , Ratones , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , ADP-Ribosa Cíclica/metabolismo , Asma/metabolismo , Contracción Muscular/fisiología , Hipersensibilidad Respiratoria/metabolismo , Fosfatidilcolinas/metabolismo , Alérgenos/metabolismo , Calcio/metabolismo , Canal Catiónico TRPA1/metabolismoRESUMEN
We previously identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of the cyclic adenosine diphosphoribose (cADPR)'s binding proteins and found that GAPDH participates in cADPR-mediated Ca2+ release from endoplasmic reticulum via ryanodine receptors (RyRs). Here, we aimed to chemically synthesise and pharmacologically characterise novel cADPR analogues. Based on the simulated cADPR-GAPDH complex structure, we performed the structure-based drug screening, identified several small chemicals with high docking scores to cADPR's binding pocket in GAPDH and showed that two of these compounds, C244 and C346, are potential cADPR antagonists. We further synthesised several analogues of C346 and found that its analogue, G42, also mobilised Ca2+ release from lysosomes. G42 alkalised lysosomal pH and inhibited autophagosome-lysosome fusion. Moreover, G42 markedly inhibited Zika virus (ZIKV, a flavivirus) or murine hepatitis virus (MHV, a ß-coronavirus) infections of host cells. These results suggest that G42 inhibits virus infection, likely by triggering lysosomal Ca2+ mobilisation and inhibiting autophagy.
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Infección por el Virus Zika , Virus Zika , Animales , Ratones , Humanos , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Virus Zika/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Autofagia , Lisosomas/metabolismo , Adenosina Difosfato Ribosa/metabolismoRESUMEN
BST-1 (bone marrow stromal cell antigen-1) is thought to be a key molecule involved in regulating the functional activity of cells in various tissues and organs. BST-1 can catalyze the hydrolysis of nicotinamide adenine dinucleotide (NAD+) to produce cyclic ADP ribose (cADPR), which activates the activity of intracellular Ca2+ signaling. Currently, the role of BST-1 regulation of Ca2+ signaling pathway in pathological myocardial hypertrophy is unclear. We found elevated expression of BST-1 in cardiac hypertrophy tissues of spontaneously hypertensive rats in our vivo study, subsequently; the mechanism of BST-1 action on myocardial hypertrophy was explored in vitro experiment. We used aldosterone (ALD) to induce H9C2 cellular hypertrophy. cADPR levels and intracellular Ca2+ concentrations declined and calcium-regulated neurophosphatase (CaN) activity and protein expression were decreased after BST-1 knockdown. And then activated T-cell nuclear factor (NFATc3) entry nucleus was inhibited. All of the above resulted in that H9C2 cells size was reduced by rhodamine-phalloidin staining. Thus, BST-1 may exacerbate cardiac hypertrophy by activating the Ca2+/CaN/NFATc3 pathway.
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Calcio , ADP-Ribosa Cíclica , Ratas , Animales , Calcio/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , ADP-Ribosa Cíclica/metabolismo , Aldosterona , Factores de Transcripción NFATC , Cardiomegalia/inducido químicamenteRESUMEN
Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.
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Antígenos CD , ADP-Ribosa Cíclica , Animales , ADP-Ribosil Ciclasa 1/genética , Antígenos CD/metabolismo , ADP-Ribosa Cíclica/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Astrocitos/metabolismo , Sinapsis/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD) is a substrate of adenosine diphosphate (ADP)-ribosyl cyclase and is catalyzed to cyclic ADP-ribose (cADPR) by CD38 and/or CD157. cADPR, a Ca2+ mobilizing second messenger, is critical in releasing oxytocin from the hypothalamus into the brain. Although NAD precursors effectively play a role in neurodegenerative disorders, muscular dystrophy, and senescence, the beneficial effects of elevating NAD by NAD precursor supplementation on brain function, especially social interaction, and whether CD38 is required in this response, has not been intensely studied. Here, we report that oral gavage administration of nicotinamide riboside, a perspective NAD precursor with high bioavailability, for 12 days did not show any suppressive or increasing effects on sociability (mouse's interest in social targets compared to non-social targets) in both CD157KO and CD38KO male mice models in a three-chamber test. CD157KO and CD38KO mice displayed no social preference (that is, more interest towards a novel mouse than a familiar one) behavior. This defect was rescued after oral gavage administration of nicotinamide riboside for 12 days in CD157KO mice, but not in CD38KO mice. Social memory was not observed in CD157KO and CD38KO mice; subsequently, nicotinamide riboside administration had no effect on social memory. Together with the results that nicotinamide riboside had essentially no or little effect on body weight during treatment in CD157KO mice, nicotinamide riboside is less harmful and has beneficial effect on defects in recovery from social behavioral, for which CD38 is required in mice.
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ADP-Ribosa Cíclica , NAD , Masculino , Ratones , Animales , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa , Ratones Noqueados , Conducta SocialRESUMEN
AIMS: Interstitial cystitis/painful bladder syndrome (IC/PBS) is characterized by lower abdominal pain and increased frequency and urgency of urine. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays role in calcium homeostasis in smooth muscle. The intracellular calcium mobilizing secondary messengers are also involved in smooth muscle contraction. The role of intracellular calcium storing depots in S1P-induced contraction was investigated in permeabilized detrusor smooth muscle having cystitis. MAIN METHODS: IC/PBS was induced by cyclophosphamide injection. The detrusor smooth muscle strips isolated from rats were permeabilized with ß-escin. KEY FINDINGS: S1P-induced contraction was increased in cystitis. S1P-induced enhanced contraction was inhibited by cyclopiazonic acid, ryanodine and heparin showing involvement of sarcoplasmic reticulum (SR) calcium stores. Inhibition of S1P-induced contraction by bafilomycin and NAADP suggested the participation of lysosome-related organelles. SIGNIFICANCE: IC/PBS triggers S1P-induced increase in intracellular calcium from SR and lysosome-related organelles in permeabilized detrusor smooth muscle.
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Cistitis Intersticial , Cistitis , Ratas , Animales , Calcio , ADP-Ribosa Cíclica , Contracción Muscular , NADP , Calcio de la DietaRESUMEN
Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are often enzymes that produce isomers of cyclic adenosine 5'-diphosphate-ribose (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR-immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate autoactive Thoeris TIRs and test the converse hypothesis: Do prokaryotic Thoeris TIRs also cross-activate plant TIR immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3'cADPR. This study demonstrates that the TIR signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a diversity of small-molecule products.
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ADP-Ribosa Cíclica , NAD+ Nucleosidasa , NAD+ Nucleosidasa/metabolismo , Receptores de Interleucina-1 , Transducción de Señal , Bacterias/metabolismo , Plantas/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD+) has emerged as a key component in prokaryotic and eukaryotic immune systems. The recent discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD+ hydrolases (NADase) links NAD+-derived small molecules with immune signaling. We investigated pathogen manipulation of host NAD+ metabolism as a virulence strategy. Using the pangenome of the model bacterial pathogen Pseudomonas syringae, we conducted a structure-based similarity search from 35,000 orthogroups for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es, including five newly identified candidates, were identified that possess domain(s) characteristic of seven NAD+-hydrolyzing enzyme families. Most Pseudomonas syringae strains that depend on the type III secretion system to cause disease, encode at least one NAD+-manipulating T3E, and many have several. We experimentally confirmed the type III-dependent secretion of a novel T3E, named HopBY, which shows structural similarity to both TIR and adenosine diphosphate ribose (ADPR) cyclase. Homologs of HopBY were predicted to be type VI effectors in diverse bacterial species, indicating potential recruitment of this activity by microbial proteins secreted during various interspecies interactions. HopBY efficiently hydrolyzes NAD+ and specifically produces 2'cADPR, which can also be produced by TIR immune receptors of plants and by other bacteria. Intriguingly, this effector promoted bacterial virulence, indicating that 2'cADPR may not be the signaling molecule that directly initiates immunity. This study highlights a host-pathogen battleground centered around NAD+ metabolism and provides insight into the NAD+-derived molecules involved in plant immunity.
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ADP-Ribosa Cíclica , NAD , Virulencia , NAD/metabolismo , ADP-Ribosa Cíclica/metabolismo , Bacterias/metabolismo , Plantas/metabolismo , Pseudomonas syringae/metabolismo , NAD+ Nucleosidasa/genética , NAD+ Nucleosidasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/microbiologíaRESUMEN
A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2'-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.
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ADP-Ribosa Cíclica , NAD , Humanos , ADP-Ribosa Cíclica/farmacología , Adenosina/farmacologíaRESUMEN
ADP-ribosyl cyclases (ADPRCs) catalyze the synthesis of the Ca2+-active second messengers Cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from NAD+ as well as nicotinic acid adenine dinucleotide phosphate (NAADP+) from NADP+. The best characterized ADPRC in mammals is CD38, a single-pass transmembrane protein with two opposite membrane orientations. The first identified form, type II CD38, is a glycosylated ectoenzyme, while type III CD38 has its active site in the cytosol. The ectoenzymatic nature of type II CD38 raised long ago the question of a topological paradox concerning the access of the intracellular NAD+ substrate to the extracellular active site and of extracellular cADPR product to its intracellular receptors, ryanodine (RyR) channels. Two different transporters, equilibrative connexin 43 (Cx43) hemichannels for NAD+ and concentrative nucleoside transporters (CNTs) for cADPR, proved to mediate cell-autonomous trafficking of both nucleotides. Here, we discussed how type II CD38, Cx43 and CNTs also play a role in mediating several paracrine processes where an ADPRC+ cell supplies a neighboring CNT-and RyR-expressing cell with cADPR. Recently, type II CD38 was shown to start an ectoenzymatic sequence of reactions from NAD+/ADPR to the strong immunosuppressant adenosine; this paracrine effect represents a major mechanism of acquired resistance of several tumors to immune checkpoint therapy.
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Fenómenos Biológicos , ADP-Ribosa Cíclica , ADP-Ribosil Ciclasa/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD/metabolismo , Conexina 43/metabolismo , ADP-Ribosa Cíclica/metabolismo , Mamíferos/metabolismo , Glicoproteínas de Membrana/metabolismo , NAD/metabolismoRESUMEN
Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.
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ADP-Ribosil Ciclasa , Proteínas Adaptadoras del Transporte Vesicular , Bacterias , Proteínas Bacterianas , ADP-Ribosa Cíclica , Inmunidad de la Planta , Receptores Toll-Like , ADP-Ribosil Ciclasa/química , ADP-Ribosil Ciclasa/genética , ADP-Ribosil Ciclasa/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADP-Ribosa Cíclica/biosíntesis , ADP-Ribosa Cíclica/química , Isomerismo , NAD/metabolismo , Dominios Proteicos , Receptores de Interleucina-1/química , Transducción de Señal , Receptores Toll-Like/química , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Triptófano/química , Triptófano/genéticaRESUMEN
Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia, IH), and it is a risk factor for cardiovascular disease (CVD) and insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of the components of Cd38-cyclic ADP-ribose (cADPR) signaling. We found that the mRNA levels of cluster of differentiation 38 (Cd38), type 2 ryanodine receptor (Ryr2), and FK506-binding protein 12.6 (Fkbp12.6) in H9c2 and P19.CL6 cardiomyocytes were significantly decreased by IH, whereas the promoter activities of these genes were not decreased. By contrast, the expression of phosphatase and tensin homolog deleted from chromosome 10 (Pten) was upregulated in IH-treated cells. The small interfering RNA for Pten (siPten) and a non-specific control RNA were introduced into the H9c2 cells. The IH-induced downregulation of Cd38, Ryr2, and Fkbp12.6 was abolished by the introduction of the siPten, but not by the control RNA. These results indicate that IH stress upregulated the Pten in cardiomyocytes, resulting in the decreased mRNA levels of Cd38, Ryr2, and Fkbp12.6, leading to the inhibition of cardiomyocyte functions in SAS patients.
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Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , ADP-Ribosil Ciclasa/genética , ADP-Ribosil Ciclasa 1 , Animales , Señalización del Calcio , Enfermedades Cardiovasculares/metabolismo , ADP-Ribosa Cíclica/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Hipoxia/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Regulación hacia ArribaRESUMEN
Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies.Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund's complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4',6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund's adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter.
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NAD , Sirtuinas , Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , ADP-Ribosa Cíclica , Humanos , Inmunoglobulina G , Leucocitos Mononucleares/metabolismo , NAD/química , NAD/metabolismo , NADP , Niacinamida , Mononucleótido de Nicotinamida , RibosaRESUMEN
Calcium (Ca2+) is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of cellular functions, such as insulin secretion from pancreatic ß-cells of the islets of Langerhans. Cyclic ADP-ribose (cADPR), synthesized from NAD+ by ADP-ribosyl cyclase family proteins, such as the mammalian cluster of differentiation 38 (CD38), is important for intracellular Ca2+ mobilization for cell functioning. cADPR induces Ca2+ release from endoplasmic reticulum via the ryanodine receptor intracellular Ca2+ channel complex, in which the FK506-binding protein 12.6 works as a cADPR-binding regulatory protein. Recently, involvements of the CD38-cADPR signal system in several human diseases and animal models have been reported. This review describes the biochemical and molecular biological basis of the CD38-cADPR signal system and the diseases caused by its abnormalities.
Asunto(s)
Antígenos CD , ADP-Ribosa Cíclica , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Calcio/metabolismo , Señalización del Calcio , ADP-Ribosa Cíclica/metabolismo , Mamíferos/metabolismo , Glicoproteínas de Membrana/metabolismoRESUMEN
Perturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.
Asunto(s)
Microbioma Gastrointestinal , Desnutrición , Animales , Bacterias/metabolismo , Niño , ADP-Ribosa Cíclica , Vida Libre de Gérmenes , Humanos , Ratones , NAD/metabolismo , NAD+ Nucleosidasa/metabolismo , Receptores de Interleucina-1RESUMEN
cADPR is a second messenger that releases Ca2+ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2'-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.