Home » Key Drug Discovery Articles » Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

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

Significance statement 

Prolyl oligopeptidase, a serine protease that cleaves short proline-containing peptides, and its inhibition by small-molecule inhibitors were under intense drug development during 1980s to early 2000, but ultimately the original hypothesis– to increase depleted neuropeptide levels in brain during aging and neurodegenerative diseases, failed. It was shown in 2008 that PREP accelerates the aggregation of α-Synuclein (aSyn) in vitro, one of the key factors in Parkinson’s disease (PD) pathology. After this finding, we studied the effects of short-term and chronic small-molecule PREP inhibitor treatment on two aged transgenic mouse strains with A30P aSyn overexpression (Thy1 and prion promoter mouse models) and in mouse strain carrying point mutation in mouse SNCA gene. Our results showed that even a short-term PREP inhibition significantly decreases the brain levels of high-molecular weight α-Synuclein oligomers, which are the most toxic species of α-Synucleinfor the cells 1,2. Since aged α-Synuclein transgenic animals have accumulated preformed α-Synuclein oligomers in their brain, we hypothesized that PREP inhibition could enhance the clearance of formed α-Synuclein oligomers. Our recent studies revealed that PREP is a negative regulator of macroautophagy (autophagy), a cellular mechanism that is critical for oligomeric aSyn clearance. Furthermore, PREP inhibition increases the levels of beclin1 protein, a positive regulator of autophagy, to enhance autophagy and the clearance of aSyn protein aggregates 2. In the present study, we show that PREP directly interacts with aSyn increasing its dimerization. Importantly, PREP inhibitor modifies the structure of PREP resulting to enhanced interaction between α-Synuclein and PREP but decreased dimerization process of α-Synuclein 3. Therefore, our current and previous results propose a dual role for PREP inhibitors to counter the toxic effects of α-Synuclein oligomers in PD.

PREP inhibition 1) reduces formation of α-Synuclein oligomers and aggregates by modulating the PREP-α-Synuclein interaction, and

2) increases autophagic clearance of α-Synuclein oligomers. Taken together, our studies suggest a promising second-coming for PREP and its inhibition as a drug development target in Parkinson’s disease.

[1] Myöhänen, TT, et al. Br.J.Pharmacol. (2012)

[2] Savolainen, MH, et al. Neurobiol.Dis. (2014)

[3] Savolainen, MH, et al. J.Biol.Chem. (2015)

Figure legend: Active forms of PREP can interact with aSyn leading to enhanced dimerization and seeding of aggregates. Inhibition of PREP with a small-molecule inhibitor, KYP-2047, stabilizes the compact form of PREP and promotes interaction with α-Synuclein, leading to decreased dimerization of α-Synuclein. PREP inhibition also overcomes the negative regulation effect of PREP on autophagy and increases the formation of autophagosomes. This enhances the clearance of α-Synuclein oligomers, leading to decreased α-Synuclein load in cells which increases cell survival.

 

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

Savolainen MH1, Yan X2, Myöhänen TT1, Huttunen HJ3. J Biol Chem. 2015 ;290(8):5117-26.

Show Affiliations

1From the Division of Pharmacology and Pharmacotherapy, University of Helsinki, FI-00014 Helsinki, Finland .

2Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland.

3Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland [email protected]

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 α-Synuclein dimerization in PREP-expressing cells but not in cells lacking PREP expression. α-Synuclein 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 α-Synuclein aggregation. Native gel analysis showed that KYP-2047 shifts PREP to a compact monomeric form with reduced ability to promote α-Synuclein nucleation. As PREP inhibition also enhances autophagic clearance of α-Synuclein, PREP inhibitors may reduce accumulation of α-Synuclein 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.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Prolyl Oligopeptidase Enhances α-Synuclein Dimerization first featured on Global Medical Discovery the World’s leading source of medical research news: featuring the innovations that will lead to a brighter tomorrow