AMPylation Regulates 5'-3' Exonuclease PLD3 processing

The 5’-3’ exonuclease phospholipase D3 (PLD3) is a type II transmembrane protein undergoing sequential post-translational modifications (PTM) by N-glycosylation, AMPylation and proteolytic cleavage. The substrates of PLD3 5’-3’ exonuclease activity are single-stranded DNAs and RNAs, which act as ligands for Toll-like receptors (TLRs) and trigger a downstream pro-inflammatory response. Although PLD3 has primarily been studied in immune cells, recent findings indicate its enrichment in neurons, where it plays a role in regulating axonal fitness in Alzheimer’s disease (AD). However, the regulatory mechanisms governing the proteolytic processing of PLD3 into its catalytically active soluble form and its functional roles in both immune and neuronal cells remain unclear. Here, we describe the functional implications of PLD3 AMPylation, its direct interaction with the protein adenylyltransferase FICD, and changes in PLD3 processing in Parkinson’s disease (PD) patient-derived neurons. We identified PLD3 AMPylation sites within the proteins’ soluble region and show that mutation of these siteslead to loss of PLD3 exonuclease catalytic activity. FICD accelerates PLD3 degradation and induces cellular stress response. Furthermore, depletion of the two human AMP-transferases FICD and SelO point towards a complex PLD3 AMPylation regulatory network in neurons. Together, our findings demonstrate a critical role of AMPylation in PLD3 processing and regulation of its catalytic activity, and provide new insights into the protein’s transport and localization to lysosomes. The observation that PLD3 regulation in PD-derived neurons is altered compared to healthy neurons further highlights its role in neurodegenerative diseases.