AMPylation Regulates 5'-3' Exonuclease PLD3 Processing

The 5'-3' exonuclease phospholipase D3 (PLD3) is a single-pass transmembrane protein undergoing sequential post-translational modifications 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 and trigger a downstream proinflammatory 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. 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 patient-derived neurons. We identified PLD3 AMPylation sites within the protein's soluble region and showed that mutation of these sites hampers PLD3 activation and its catalytic activity. Overexpression of FICD AMP transferase accelerates PLD3 degradation and induces cellular stress response. 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 Parkinson's disease-derived neurons is altered compared with healthy neurons further highlights its role in neurodegenerative diseases.