Elucidation of the interaction network of diadenosine triphosphate

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Cells contain several minor nucleotides in addition to the canonical mono-, di- and triphosphates. Among these, various dinucleoside polyphosphates (NpnN, n = 2-7) are found. The dinucleoside polyphosphates consist of two nucleoside moieties, which are linked by a polyphosphate chain at the respective 5'-hydroxyl groups. These molecules can be found in prokaryotic as well as eukaryotic cells. Despite being known for 50 years the functions of dinucleoside polyphosphates are still unclear. One of the most studied one is diadenosine triphosphate (Ap3A). Its cellular concentrations vary dependent on cell type and environmental factors in a nanomolar to a lower micromolar range. An increase of Ap3A concentration was reported several times upon stress conditions. Therefore it was postulated, that Ap3A serve as a cellular signal (alarmones) of stressors and thus be involved in the adaptive processes of cells to stress conditions. However, their exact role is yet to be specified. The known Ap3A hydrolase in humans, and sole verified interaction partner, is the fragile histidine triad protein (Fhit). It is encoded by the FHIT gene, one of the most fragile sites of the human genome. Hence, the loss of Fhit expression and the altering of its enzymatic activity are commonly observed in human cancer cells. The Fhit-Ap3A complex is suggested to be the active molecule for tumor suppressor activity without knowing further interaction partners. Since the knowledge about the Ap3A interactome is sparse we aim at developing and applying new means for the discovery of Ap3A interaction partners within this project. We will develop and synthesize non-hydrolysable Ap3A (nh Ap3A) analogues that bear a light sensitive functionality for photoaffinity crosslinking and an affinity tag. Upon incubation with cell lysates and irradiation, the photoreactive nh Ap3A analogue will covalently bind to interacting proteins, which in turn will be labelled with the affinity tag. After enrichment of the labelled proteins we will conduct affinity purification and the proteins will be identified by mass spectrometry (MS). Hereby, we will closely cooperate with the Proteomics Facility of the University of Konstanz. Thereupon, we will confirm the identified proteins ability to interact with Ap3A. Furthermore, we aim at gaining insights into the interactome of Fhit, the hydrolase of Ap3A in humans, and Fhit in complex with Ap3A. Therefore, we will device means to covalently connect an nh Ap3A analogue to the active site of Fhit. The Fhit-Ap3A complex will be employed for the formation of affinity matrices for the identification of selective binding partners by affinity purification. Since these experiments will be conducted in parallel using Fhit- and Fhit-Ap3A complex-modified matrices insights into the Fhit and Fhit-Ap3A interactome will be obtained. Again, proteins will be identified my MS and their interaction with Fhit and Fhit-Ap3A will be studied.