Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases.

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

MS-Based in Situ Proteomics Reveals AMPylation of Host Proteins during Bacterial Infection.

Bacteria utilize versatile strategies to propagate infections within human cells, e.g., by the injection of effector proteins, which alter crucial signaling pathways. One class of such virulence-associated proteins is involved in the AMPylation of eukaryotic Rho GTPases with devastating effects on viability. In order to get an inventory of AMPylated proteins, several technologies have been developed. However, as they were designed for the analysis of cell lysates, knowledge about AMPylation targets in living cells is largely lacking. Here, we implement a chemical-proteomic method for deciphering AMPylated host proteins in situ during bacterial infection. HeLa cells treated with a previously established cell permeable pronucleotide probe (pro-N6pA) were infected with Vibrio parahaemolyticus, and modified host proteins were identified upon probe enrichment and LC-MS/MS analysis. Three already known targets of the AMPylator VopS-Rac1, RhoA, and Cdc42-could be confirmed, and several other Rho GTPases were additionally identified. These hits were validated in comparative studies with V. parahaemolyticus wild type and a mutant producing an inactive VopS (H348A). The method further allowed to decipher the sites of modification and facilitated a time-dependent analysis of AMPylation during infection. Overall, the methodology provides a reliable detection of host AMPylation in situ and thus a versatile tool in monitoring infection processes.