Design and synthesis of cyclic ADP-4-thioribose as a stable equivalent of cyclic ADP-ribose, a calcium ion-mobilizing second messenger.
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ABSTRACT:
SUBMITTER: Tsuzuki T
PROVIDER: S-EPMC3738939 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approach to design drug-like signalling pathway modulators.
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Project description:The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.
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Project description:Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca(2+) concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.
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Project description:Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.
| S-EPMC2888401 | biostudies-literature
Project description:Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 microg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD+ levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]i), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs.
| S-EPMC1223684 | biostudies-other
Project description:Cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A?+?L196F?+?C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the 'northern' ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the 'northern' ribose. First, the mutant F37A?+?L196F?+?C253G, with a smaller residue 253 (Ala?>?Gly), showed increased cADPR specificity. Then, the mutant F37A?+?L196F?+?V252A?+?C253G, with another residue made smaller (Val?>?Ala), displayed the desired specificity, with cADPR kcat/KM ?20-200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.
| S-EPMC5773619 | biostudies-literature
Project description:Cyclic ADP ribose (cADPR) is a potent Ca(2+)-releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. beta-NAD+, cGMP and nitric oxide (NO) have been reported to mobilize Ca2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca(2+)-mobilizing action of beta-NAD+, cGMP and NO, but has no effect on cADPR-induced Ca2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca2+, we have shown that nicotinamide inhibits cADPR-mediated Ca2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca2+ release by beta-NAD+, cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.
| S-EPMC1217811 | biostudies-other
Project description:The multifunctional, transmembrane glycoprotein human CD38 catalyses the synthesis of three key Ca2+-mobilising messengers, including cyclic adenosine 5'-diphosphate ribose (cADPR), and CD38 knockout studies have revealed the relevance of the related signalling pathways to disease. To generate inhibitors of CD38 by total synthesis, analogues based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesised. In the first example of a sugar hybrid cIDPR analogue, "L-cIDPR", the natural "northern" N1-linked D-ribose of cADPR was replaced by L-ribose. L-cIDPR is surprisingly still hydrolysed by CD38, whereas 8-Br-L-cIDPR is not cleaved, even at high enzyme concentrations. Thus, the inhibitory activity of L-cIDPR analogues appears to depend upon substitution of the base at C-8; 8-Br-L-cIDPR and 8-NH2-L-cIDPR inhibit CD38-mediated cADPR hydrolysis (IC50 7 μM and 21 µM respectively) with 8-Br-L-cIDPR over 20-fold more potent than 8-Br-cIDPR. In contrast, L-cIDPR displays a comparative 75-fold reduction in activity, but is only ca 2-fold less potent than cIDPR itself. Molecular modelling was used to explore the interaction of the CD38 catalytic residue Glu-226 with the "northern" ribose. We propose that Glu226 still acts as the catalytic residue even for an L-sugar substrate. 8-Br-L-cIDPR potentially binds non-productively in an upside-down fashion. Results highlight the key role of the "northern" ribose in the interaction of cADPR with CD38.
| S-EPMC5700923 | biostudies-literature
Project description:Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2'F-2'deoxynicotinamide adenine dinucleotide (ribo-2'-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.
| S-EPMC2785691 | biostudies-literature
Project description:Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.
| S-EPMC4334965 | biostudies-literature