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Cyclic-di-GMP cyclic di-guanylate

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Cyclic-di-GMP cyclic di-guanylate

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  1. BiophosphatesCyclic nucleotides: cAMP, cGMP(well known second messengers)c-di-GMP (recently identified global bacterial second messenger)Phospholipids (structural function) vsLysophospholipids (ligands in signal transduction)Mononucleotides (NTP, dNTP)Mononucleotides (ATP, UTP, UDP, ADP) as ligands in signal transduction

  2. Cyclic-di-GMPcyclic di-guanylate

  3. Structure of the cyclic-diguanylic acid(cyclic di-GMP)

  4. Allosteric regulation of cellulose synthase by cyclic bis(3’-5’)diguanylic acidP. Ross et al., Microbiological Reviews, 1991, vol. 55, 35-58.

  5. Synthesis and degradation of cyclic di-GMP, based on studies with G. xylinus (Ross et al., 1991).Synthesis of c-di-GMP from two molecules of GTP occurs in two steps (with pppGpG as intermediate). Degradation of c-di-GMP is also two-step process, with a linear dinucleotide (pGpG) intermediate, which, finally, is degraded to guanosine 5’-monophosphate.

  6. Enzymes responsible for c-di-GMP metabolism: diguanylate cyclase (DGC) and phosphodiesterase A1: Bacterial proteins possessing diguanylate cyclase (DGC) activity contain GGDEF domain (glycine-glycine-aspartate-glutamate-phenylalanine), which can bind GTP. This domain catalyzes the formation of c-di-GMP. Phosphodiesterase degrading c-di-GMP possesses both GGDEF and EAL (glutamate-alanine-leucine) domains. In this case GGDEF domain can also bind GTP but functions as regulatory domain for phosphodiesterase activity. It means that the phosphodiesterase A1 is allosterically controlled by the cellular GTP level.

  7. Phosphodiesterase A1 is also regulated by O2 concentration: it is a a heme-based sensor of O2 concentration Under anaerobic conditions the enzyme is most active; it means that cellulose production is low in O2-depleted culture of G. xylinus. A.L. Chang et al., Biochemistry, 2001, 40: 3420-3426.

  8. Cyclic diguanylate: second messenger in bacteria Known for more than 15 years as an activator of cellulose synthase in Gluconacetobacter xylinus, c-di-GMP is emerging as a novel pleiotropic second messenger in bacteria. The GGDEF- and EAL-containing proteins involved in c-di-GMP synthesis and degradation, are (almost) ubiquitous in bacterial genomes. These proteins affect cell differentiation and multicellular behaviour as well as interactions between the microorganisms and their eukaryotic hosts. U. Jenal: Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? Curr Opin Microbiol. 2004, 7:185-91. U. Romling et al.: C-di-GMP: the dawning of a novel bacterial signalling system. Mol. Microbiol. 2005, 57: 629-639.

  9. c-di-GMP-dependent signaling pathways are considered as potential targets for new antimicrobial agents c-di-GMP is also responsible for a formation of biofilm, which covers a surface of many pathogenic bacteria (e.g. Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Salmonella typhimurium). For this reason, the biofilm formation can be influenced by phosphodiesterase A1 activity and the level of c-di-GMP A.D. Tischler et al.: Infect Immun. 2005, 73: 5873-82. Cyclic diguanylate regulates Vibrio cholerae virulence gene expression.

  10. The Wsp chemosensory pathway in Pseudomonas In this model, WspE kinase activity is activated via signal-dependent conformational changes in the WspA/WspB or WspA/WspD complex. WspE phosphorylates WspR leading to downstream signal propagation. Intracellular information transfer is indicated by arrows.

  11. Lysophospholipids Phospholipids

  12. Lysophospholipids have been recently discovered as ligands activating specific membrane receptors coupled with G proteins Production of bioactive LPA by autotaxin/lysoPLD. Soluble ATX/lysoPLD hydrolyzes carrier-bound and membrane-associated LPC (and other lysophospholipids) to generate LPA. Newly produced LPA acts on its own G protein–coupled receptors and thereby evokes numerous cellular responses, including Ras-mediated cell proliferation and Rho/Rac-regulated cell migration. Excess LPA is converted into monoacylglycerol (MAG) by membrane-bound lipid phosphatases.

  13. Biological roles of lysophospholipids in different systems: cellular responses to LPA and S1P such as survival, proliferation and migration are particularly important within the nervous system, the cardiovascular system, the immune system and female reproductive system.

  14. Lysophosphatidic acid (LPA) signaling mechanisms. LPA interacts with G-protein-coupled receptors (LPA1-3) to regulate pathways that are coupled to Rho family GTPases, MAP kinase activation and, ultimately, control of cell migration, proliferation, survival and differentiation. Interaction of LPA with intracellular PPAR-γ directly regulates nuclear gene transcription. LPPs negatively regulate LPA signaling by degrading LPA at the membrane surface. LPA1–3, LPA receptors 1–3; LPPs, lipid phosphate phosphatases; MAP kinase, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PPAR-γ, peroxisome-proliferator-activated receptor γ.

  15. Structure of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family members. Domain structures of ENPP1–5 highlight the conserved nuclease (NUC), phosphodiesterase (PDE),somatomedin and transmembrane domains. Red arrows identify possible proteolytic processing sites that would result in release of the soluble catalytic domain from the integral membrane precursor.

  16. ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family members

  17. ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family members

  18. FTY720 is tested as an immunomodulator for the use after transplantation and for a treatment of multiple sclerosis

  19. Mononucleotides as receptor ligands

  20. Agonists of nucleotide receptors

  21. Antagonists of nucleotide receptors

  22. Agonists of P2Y2 nucleotide receptors used in clinical studies as agents for cystic fibrosis treatment

  23. AA number Agonist P2X1 399 ATP, UTP P2X2 472, 401 ATP, UTP P2X3 397 ATP, UTP P2X4 388, 329 ATP P2X5 455 ATP P2X6 379 ATP P2X7 595 ATP List of known nucleotide receptors and their agonists AA number Agonist P2Y1 362 ADP, ATP P2Y2 373 ATP, UTP P2Y4 352 UTP P2Y6 379 UDP P2Y11 371 ATP P2Y12 342 ADP P2Y13 ADP P2Y14 UDP-glucose

  24. Future perspectives Receptor Agonist or antagonist Application ------------------------------------------------------------------------------------------------- P2Y2 UTP, Up4U, Cp4U cystic fibrosis treatment P2Y2 UTP, Up4U, Cp4U dry eye syndrom P2Y12 clopidogrel, ticlopidine inhibition of thrombosis P2Y4, P2Y6 ??? Wound healing P2X, P2Y6 ??? Pain treatment How to achieve these goals? 1. Identification of the P2 receptors on cell membranes of different cell types; 2. Studies on structure-activity relationships of P2 receptors; 3. Chemical synthesis of new specific agonists and antagonists; 4. Pharmacological studies with the use of specific antagonists.

  25. Extracellular nucleotides can activate P2X and/or P2Y receptors, but they are also substrates for ecto-enzymes

  26. Extracellular nucleotides can activate P2X and/or P2Y receptors, but they are also substrates for ecto-enzymes

  27. Novel inhibitory compounds target the production and action of LPA and S1P. cPA inhibits ATX which catalyzes the extracellular production of LPA from LPC.LPA can then bind to the LPA receptors at the cell surface and stimulate intracellular signaling cascades. Lipid phosphate phosphastases (LPP), integral membrane proteins with the catalytic site facing out, in the plasma membrane hydrolyze the phosphate from LPA to yield monoacylglycerol (MAG).The brown recluse spider venom produces LPA fromLPC and also ceramide 1-phosphate from sphingomyelin. Sphk phosphorylates sphingosine or its analogue, FTY720, intoS1P or phospho-FTY720, respectively, which bind S1P receptors. Sphingomab neutralizes S1P by binding to it andpreventing receptor activation.The chemicalstructures represented are typical structures and it is important to note that several species with different hydrophobic chains exist. Here is the natural 1-oleoyl speciesfor LPC, LPA, and monoacylglycerol. N-stearoyl species are shown for sphingomyelin and ceramide 1-phosphate.

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