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 Ap(4)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 Ap(4)A levels during cellular stress differs from when Ap(4)A is present at low concentrations, allowing response to extracellular cues.

Automated summary

Diadenosine polyphosphates (Ap(n)As) are non-canonical nucleotides that increase in cellular concentrations during stress and act as alarmones to regulate homeostasis. This study investigates the role of Ap(n)As in protein AMPylation, a post-translational modification that transfers adenosine monophosphate (AMP) to proteins. The findings demonstrate that Ap(4)A can be efficiently utilized for AMPylation by FICD and identify potential new AMPylation targets. Interestingly, the study suggests that the AMPylation targets of FICD may vary depending on the nucleotide cosubstrate.