Abstract
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.
Highlights
Prolyl oligopeptidase (PREP) modulates accumulation of aggregated ␣-synuclein both in vitro and in vivo, but the mechanism remains unknown
It was previously shown that PREP accelerates aggregation of ␣-synuclein (aSyn) aggregation and that a PREP inhibitor blocks this in vitro and promotes clearance of aSyn aggregates in aSyn-overexpressing cells and in vivo models [14, 22]
In the current paper we have shown that PREP directly interacts with aSyn and increases its dimerization and that this effect can be countered with a specific PREP inhibitor, KYP-2047
Summary
Prolyl oligopeptidase (PREP) modulates accumulation of aggregated ␣-synuclein both in vitro and in vivo, but the mechanism remains unknown. We have identified PREP as a negative modulator of autophagosome formation, and treatment with PREP inhibitor, KYP-2047, induced macroautophagy and autophagosome formation via a beclin 1-dependent pathway [23] This was accompanied with increased clearance of high molecular weight aSyn species in the A30P transgenic mouse brain, suggesting PREP inhibition as a novel therapeutic strategy in synucleinopathies [23]. We have used a combination of live-cell and cell-free methods to show that both wild-type PREP and a catalytically inactive mutant PREP with a serine-alanine mutation at residue 554 (S554A) interact with aSyn with low micromolar affinity Both wild-type PREP and the S554A mutant enhance aSyn dimerization in cells, but only in cells expressing wild-type PREP is aSyn dimerization reduced by KYP-2047, a specific PREP inhibitor. These data provide further support for PREP as a nucleation point for aSyn aggregation and provide mechanistic insight in how pharmacological inhibition of PREP reduces aSyn aggregation in cells
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
