guanosine-triphosphate and diadenosine-tetraphosphate

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

Topics: Bacillus subtilis; Dinucleoside Phosphates; Guanosine Triphosphate

The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca(2+)] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y(4) receptor, UTP, GTP, and ITP all increased intracellular [Ca(2+)] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC(50), microM). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A), all at 100 microM, were inactive at the hP2Y(4) receptor. In cells expressing the rP2Y(4) receptor, all seven nucleotides increased intracellular [Ca(2+)] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0. 51) > Ap(4)A (1.24) approximately ITP (1.82) approximately GTP (2. 28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K(B) = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 microM) inhibited the response to an EC(50) concentration of UTP by approximately 40 and approximately 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Dinucleoside Phosphates; Humans; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Species Specificity; Tumor Cells, Cultured; Uridine Diphosphate

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

Topics: Animals; Binding Sites; Binding, Competitive; Cell Membrane; Dinucleoside Phosphates; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Lung; Radioligand Assay; Rats; Receptors, Cell Surface; Sulfur Radioisotopes; Tritium

As part of the exploration of the mechanism of platinum(II) complex-induced growth inhibition and/or cytotoxicity, we studied the intracellular levels of several nucleotides during treatment of mouse leukemia P388/D1 at selected concentrations of 1 microM cis-diamminedichloroplatinum(II) (cis-DDP) and 20 microM of its trans-isomer (trans-DDP). The effects and their time-dependences are correlated with those on cell growth parameters previously published (Just G and Holler E, Cancer Res 49: 7072-7078, 1989). The effects of cis-DDP are strong and irreversible, whereas those of trans-DDP are marginal and reversible, in parallel with similar effects on cell growth parameters. Concentrations of nucleoside 5'-di- and 5'-triphosphates increase in parallel with cellular DNA and protein content by three- to four-fold after 60 hr of treatment. The nucleoside monophosphates dAMP, dGMP and dTMP reveal concentration maxima during exponential cell growth that are two- to six-fold higher than in the control cultures. Levels of cyclic AMP, adenosine(5')tetraphospho(5')adenosine (Ap4A) and CDP rise three- to five-fold above those in the control cultures within a few hours of the start of treatment. The level of coenzyme NAD+ falls below that of the control, concomitantly with an arrest of cells in the G2 phase of the cell cycle and with the appearance of giant cells. Due to the high reactivity of cis-DDP and the continuous concentration increase during the treatment, purine nucleoside 5'-triphosphates provide a possibility for the acquisition of resistance to cis-DDP. The correlation of responses of metabolically and regulatory active nucleotides with biological effects suggests their function in antitumorigenesis.

Topics: Adenosine Triphosphate; Animals; Cisplatin; Cyclic AMP; Cytidine Diphosphate; Dinucleoside Phosphates; DNA; Guanosine Triphosphate; Kinetics; Leukemia P388; Mice; Proteins; Ribonucleotides; Tumor Cells, Cultured