The aggregation of α-synuclein (α-Syn) is a characteristic of Parkinson’s disease (PD). α-Syn oligomerization/aggregation is accelerated by the serine peptidase, prolyl oligopeptidase (POP). Factors that affect POP conformation, including most of its inhibitors and an impairing mutation in its active site, influence the acceleration of α-Syn aggregation resulting from the interaction of these proteins. It is noteworthy, however, that α-Syn is not cleaved by POP. Prolyl endopeptidase-like (PREPL) protein is structurally related to the serine peptidases belonging to the POP family. Based on the α-Syn–POP studies and knowing that PREPL may contribute to the regulation of synaptic vesicle exocytosis, when this protein can encounter α-Syn, we investigated the α-Syn–PREPL interaction. The binding of these two human proteins was observed with an apparent affinity constant of about 5.7 μM and, as in the α-Syn assays with POP, the presence of PREPL accelerated the oligomerization/aggregation events, with no α-Syn cleavage. Furthermore, despite this lack of hydrolytic cleavage, the serine peptidase active site inhibitor phenylmethylsulfonyl fluoride (PMSF) abolished the enhancement of the α-Syn aggregation by PREPL. Therefore, given the attention to POP inhibitors as potential drugs to treat synucleinopathies, the present data point to PREPL as another potential target to be explored for this purpose.

We describe a method tailored to the in-cell selection of protease inhibitors. In this method, a target protease is co-expressed with a selective substrate, the product of which kills host cells. Therefore, the method can be applied to identify potential inhibitors based on cell host survival when inhibition of the target protease occurs. The TEV protease was chosen for this proof-of-concept experiment. The genetically encoded selective substrate is a single polypeptide chain composed of three parts: (1) a ccdB protein, which can cause host cell death when it accumulates inside the cell; (2) a protease cleavage sequence that can be changed according to the target protease, in this case the TEV substrate ENLYFQ↓G (↓-predicted cleavage site); and (3) the ssrA sequence (AANDENYALAA), which drives the polypeptide to degradation by the ClpX/ClpP complex inside host E. coli cells. In our experiment, co-expression of the active TEV protease and this selective substrate (ccdB-ENLYFQG-ssrA) caused the death of a significant host cell population, while control assays with an inactive mutant TEV Asp81Asn did not. Details of the methodology used are given, providing the basis for the application of similar systems for other proteases of interest.