Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown. We have now used protein-fragment complementation assays (PCA) and microscale thermophoresis in parallel to show that PREP interacts directly with aSyn in both intact cells and in a cell-free system. Using split luciferase-based PCA, we first showed that PREP enhances the formation of soluble aSyn dimers in live Neuro-2A neuroblastoma cells. A PREP inhibitor, KYP-2047, reduced aSyn dimerization in PREP-expressing cells but not in cells lacking PREP expression. aSyn dimerization was also enhanced by PREP(S554A), an enzymatically inactive PREP mutant, but this was not affected by KYP-2047. PCA and microscale thermophoresis studies showed that aSyn interacts with both PREP and PREP(S554A) with low micromolar affinity. Neither the proline-rich, C-terminal domain of aSyn nor the hydrolytic activity of PREP was required for the interaction with PREP. Our results show that PREP binds directly to aSyn to enhance its dimerization and may thus serve as a nucleation point for aSyn aggregation. Native gel analysis showed that KYP-2047 shifts PREP to a compact monomeric form with reduced ability to promote aSyn nucleation. As PREP inhibition also enhances autophagic clearance of aSyn, PREP inhibitors may reduce accumulation of aSyn inclusions via a dual mechanism and are thus a novel therapeutic candidate for synucleinopathies. Our results also suggest that PREP has other cellular functions in addition to its peptidase activity. Background: Prolyl oligopeptidase (PREP) modulates accumulation of aggregated α-synuclein both in vitro and in vivo, but the mechanism remains unknown. Results: Using live-cell and cell-free methods, we show that PREP interacts directly with α-synuclein. Conclusion: PREP binds to α-synuclein and enhances its dimerization. Significance: These results establish a mechanism of how PREP inhibition reduces α-synuclein aggregation and support PREP inhibition as a novel therapy in synucleinopathies.