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The products of ATP consuming metabolic pathways, which we discovered in marine sponges, may be considered as signal-transducing molecules. The first stage of the first pathway, the synthesis of 2',5'-oligoadenylates (2-5A), is catalysed by the enzyme different from any other known (vertebrate) 2-5A synthetases as it does not require dsRNA for its activation. While in mammals the signal is transduced via 2-5A from interferon to the only known target protein, latent endoribonuclease, then in sponges the signal pathway remains unknown. One of the primary tasks of this project is to identify and characterise the protein(s) regulated by 2-5A in the sponge both on the protein and gene level. The characterisation of the complete 2-5A system in sponges as the result of further studies of different sponge species will answer the question about its functions in sponges and its potential evolutionary development: it is still to be established how close these functions can be to the presently known role(s) of the relevant mammalian systems. In order to answer this question, we plan to study the 2-5A synthetase in a new model organism for evolutionary biology, in the ascidian Ciona intestinalis. Our initial working hypothesis is the role of 2-5A as a signal molecule, participating in the organism defence function. The other, ATP-converting unique nucleosidase activity discovered by us could also participate in the signal transduction via its reaction product, adenine. In this study we plan to isolate pure ATP N-glycosidase from the marine sponge Axinella polypoides (and possibly from a freshwater sponge) and then define its gene and genomic structure. The analysis of the deduced amino acid sequence would indicate the position of the novel enzyme in the classification of enzymes (or proteins in general) and to find its potential relatives in other organisms (including human). We plan to establish a freshwater sponge culture in our lab, in order to enhance research (including the adenine and adenosine nucleotide receptor studies on sponge cells). Our final aim in ATP N-glycosidase studies is to define its natural function. The enzyme seemingly defies the basic principles of nucleotide metabolism and has unusually high specific activity. In this project we also hope to answer the question arisen from our previous experimental work: whether ATP-conversion in sponge cells to 2-5A from one side, and ATP direct degradation into adenine, catalysed by ATP N-glycosidase (together with ADP-ribosyl cyclase pathway) from the other side, are mutually exclusive alternative pathways of ATP-utilisation or they can simultaneously exist in the same cells. |