The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation

Diadenosine polyphosphates (Ap_n As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Ap_n As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap₄ A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap₄ A levels during cellular stress differs from when Ap₄ A is present at low concentrations, allowing response to extracellular cues.