Formation of the Alarmones Diadenosine Triphosphate and Tetraphosphate by Ubiquitin- and Ubiquitin-like-Activating Enzymes

Diadenosine polyphosphates (Ap(n)As) such as diade-nosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular Ap(n)A concentrations increase, it was postulated that Ap(n)As are alarmones triggering stress-adaptive processes. The major synthesis pathway of Ap(n)As is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like Ap(n)A formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to Ap(n)A formation for the first time.