The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.

Funding information Deutsche ForschungsgemeinschaftIntracellular dinucleoside polyphosphates (Np n Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap 3 A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap 3 A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap 3 A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap 3 A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap 3 A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit.

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