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Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems.

ABSTRACT: We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.

SUBMITTER: Chini EN

PROVIDER: S-EPMC1219808 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by ?-Adrenergic Stimulation.

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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.

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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.

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Project description:Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.

| S-EPMC10175808 | biostudies-literature

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