The prolyl oligopeptidase and α-synuclein connection revisited.

Abstract

The aim of this work was to revisit the connection between prolyl oligopeptidase (PREP) and α-synuclein (aSyn) by presenting novel data from cell free and cellular assays and to discuss the results in a contemporary context. The aSyn aggregation process was studied using fluorescence correlation spectroscopy and thioflavin-T fluorescence. Binding sites for PREP on the aSyn sequence were determined using peptide arrays. Subcellular localisation of PREP and stress markers were studied using double staining immunofluorescence microscopy in SH-SY5Y cells with and without overexpression of aSyn and PREP, before and after differentiation, and with or without proteolytic stress induced by proteasome inhibition. The interaction between PREP and aSyn was found to be weak and transient. It promotes the early phases of aggregation but does not affect the rate of β-fibril formation. Moreover, this interaction is not dependent upon the C-terminal prolines of aSyn, but is affected by PREP inhibitors and interferes with PREP substrate binding. Although present in the same cellular compartments, there is little evidence for a strong physical association of PREP with aggresomes and stress markers. Instead, there is colocalization with aSyn in the cell periphery and neurites. There is evidence for a binding site for peptides much longer than the usual PREP substrates. The modular assembly of molecular machines and the observation that PREP's protein-protein interactions are tuneable by active site inhibitors, lead to the hypothesis that this binding site features in the cross-talk between autophagy and neuron-specific pathways involving vesicle transport and protein secretion.