Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

The nucleotides diadenosine triphosphate (Ap(3)A) and diadenosine tetraphosphate (Ap(4)A) 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 Ap(3)A and Ap(4)A 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 Ap(n)A 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 Ap(n)As across the domains of life, but also identify those that are different for Ap(3)A or Ap(4)A. 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.