BACKGROUND: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. METHODS: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. RESULTS: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. CONCLUSION: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.
The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 A resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.
Most kinetic studies of prolyl oligopeptidase (PREP) were performed with the porcine enzyme using modified peptide substrates. Yet recent biophysical studies used the human homolog. Therefore, the aim of this study was to compare the kinetic behavior of human and porcine PREP, as well as to find a suitable method to study enzyme kinetics with an unmodified biological substrate. It was found that human PREP behaves identically to the porcine homolog, displaying a double bell-shaped pH profile and a pH-dependent solvent kinetic isotope effect of the kcat/Km, features that set it apart from the related exopeptidase dipeptidyl peptidase IV (DPP IV). However, the empirical temperature coefficient Q10, describing the temperature dependency of the kinetic parameters and the non-linear Arrhenius plot of kcat/Km are common characteristics between PREP and DPP IV. The results also demonstrate the feasibility of microcalorimetry for measuring turn-over of proline containing peptides.
Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM.
Prolyl oligopeptidase (PREP, E.C.3.4.21.26) is a cytosolic serine protease that hydrolyzes small (<3 kDa), proline-containing peptides on the carboxyl terminal side of proline residues, and is widely distributed in the brain. High PREP activity, due to aging or neurodegenerative disease, has been hypothesised to lead to an increased breakdown of neuropeptides, resulting in a decline of cognitive functions and an acceleration of neurodegeneration. Recent data have suggested that PREP involvement in neurodegeneration cannot be explained by its extracellular space proteolytic activity alone, but may involve intracellular PREP activities as well. In order to test this, appropriate methods for measuring PREP intracellular activity must first be developed. In the present study, we developed and validated an in situ PREP intracellular activity assay in primary rat cortical neurons, using nitroblue tetrazolium chloride salt (NBT) and a PREP specific substrate (S)-benzyl 2-(2-(4-hydroxynaphthalen-l-ylcarbanoyl)pyrrolidin-l-yl)-2-oxoethylcarbamate (UAMC-00682). This novel in situ PREP activity assay was further validated in neuroblastoma SH-SY5Y cells, under conditions of PREP overexpression and inhibited PREP expression. Using this assay, we demonstrated that PREP inhibitors, Z-Pro-Pro-aldehyde-dimethylacetal, Boc-Asn-Phe-Pro-aldehyde, and (S)-1-((S)-1-(4-phenylbutanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (KYP-2047), were able to inhibit intracellular PREP activity in primary rat cortical neurons. KYP-2047 was the most potent PREP inhibitor in all assay systems tested. The validated assay enables localization and quantification of in situ PREP activity in primary rat cortical neurons and neuroblastoma SH-SY5Y cells, as well allows testing cell permeability and efficiency of novel PREP inhibitors.
BACKGROUND AND PURPOSE: The aggregation of alpha-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of alpha-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on alpha-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human alpha-syn and in the brains of two A30P alpha-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 x 3 mg.kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble alpha-synuclein protein levels were measured by Western blot. alpha-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and alpha-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of alpha-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with alpha-synuclein inclusions while abolishing the colocalization of alpha-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated alpha-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of alpha-synuclein immunoreactivity and soluble alpha-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of alpha-synuclein, while KYP-2047 seems to effectively prevent these processes.
We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on alpha-synuclein.
Title: Structure and function relationship in prolyl oligopeptidase Van Elzen R, Lambeir AM Ref: CNS Neurol Disord Drug Targets, 10:297, 2011 : PubMed
Prolyl oligopeptidase (POP) belongs to a unique class of serine proteases. Based on extensive enzyme kinetic measurements it has become clear that POP acts in a multifaceted way. This is reflected in the complex behavior in different reaction conditions with different substrates. Also the typical structural architecture of POP, with the active site located at the interface of the catalytic domain and the beta-propeller domain, has instigated many researchers to speculate about the mechanism of functioning. The latest developments support the idea of extended conformational changes upon substrate binding. In this review the plethora of available information is assembled into a coherent and stepwise description of the structural composition of POP. In one aspect the composition and contribution of structural boundaries at the active site are described. Attention is focused on the catalytic components and the features that are presumed to confine the substrate specificity. Complementary to this, the specificity of POP towards the residues surrounding the scissile bond is described by means of a consecutive evaluation of the preferred physico-chemical properties. Together, these two approaches may facilitate a better understanding of the concepts that determine catalytic behavior of POP in physiological conditions.
