Prolyl oligopeptidase inhibition reduces PolyQ aggregation and improves cell viability in cellular model of Huntington's disease.
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ABSTRACT:
SUBMITTER: Norrbacka S
PROVIDER: S-EPMC6850970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND AND PURPOSE: The aggregation of ?-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of ?-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on ?-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human ?-syn and in the brains of two A30P ?-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 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble ?-synuclein protein levels were measured by Western blot. ?-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and ?-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 ?-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with ?-synuclein inclusions while abolishing the colocalization of ?-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated ?-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 ?-synuclein immunoreactivity and soluble ?-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of ?-synuclein, while KYP-2047 seems to effectively prevent these processes.
| S-EPMC3417432 | biostudies-literature
Project description:Prolyl oligopeptidase (PREP) inhibition by small-molecule inhibitors can reduce alpha-synuclein (aSyn) aggregation, a key player in Parkinson's disease pathology. However, the significance of PREP protein for aSyn aggregation and toxicity is not known. We studied this in vivo by using PREP knock-out mice with viral vector injections of aSyn and PREP. Animal behavior was studied by locomotor activity and cylinder tests, microdialysis and HPLC were used to analyze dopamine levels, and different aSyn forms and loss of dopaminergic neurons were studied by immunostainings. Additionally, PREP knock-out cells were used to characterize the impact of PREP and aSyn on autophagy, proteasomal system and aSyn secretion. PREP knock-out animals were nonresponsive to aSyn-induced unilateral toxicity but combination of PREP and aSyn injections increased aSyn toxicity. Phosphorylated p129, proteinase K resistant aSyn levels and tyrosine hydroxylase positive cells were decreased in aSyn and PREP injected knock-out animals. These changes were accompanied by altered dopamine metabolite levels. PREP knock-out cells showed reduced response to aSyn, while cells were restored to wild-type cell levels after PREP overexpression. Taken together, our data suggests that PREP can enhance aSyn toxicity in vivo.
| S-EPMC5784134 | biostudies-literature
Project description:BackgroundProlyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis.MethodsThe human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor) at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot.ResultsCompared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 ?M) incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA)-treated cells at 26-130 ?M; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026-65 ?M without FFA treatment. S17092 treatment (13 and 26 ?M) also elicited no significant effect on apoptosis in normal L02 cells, but FFA treatment increased cell apoptosis, which was attenuated by S17092 incubation. S17092 treatment inhibited intracellular POP activity and decreased the AcSDKP level at the concentration of 0.026-26 ?M. After treatment with FFA for 24 h, oil red O staining revealed significant lipid accumulation in the cells in the model group compared with the controls; however, lipid accumulation was suppressed after the administration of S17092 (13 and 26 ?M). Accordingly, the triglyceride levels in the FFA-treated cells were approximately 5-fold greater than those of the controls and were decreased by approximately 25% and 45% after the administration of S17092 at 13 and 26 ?M, respectively. The mRNA levels of FASN, PPAR-?, and SREBP-1c were higher in the FFA-treated cells than in the normal controls, and all of these levels were significantly inhibited in the presence of S17092 at both 13 and 26 ?M. S17092 treatment did not affect LC3B II in the FFA-treated cells compared with FFA treatment alone.ConclusionThe expression of POP increases with hepatocyte steatosis, and POP inhibitors can significantly reduce intracellular lipid accumulation, which might be related to the inhibition of genes involved in lipid synthesis.
| S-EPMC5070736 | biostudies-literature
Project description:Glioblastoma (GB) is the most aggressive tumor of the central nervous system (CNS), characterized by excessive proliferation, necrosis and invasiveness. The survival rate for patients with GB still remains low. Angiogenesis and apoptosis play a key role in the development of GB. Thus, the modulation of angiogenesis and apoptosis processes represent a possible strategy to counteract GB progression. This study aimed to investigate the potential effect of KYP-2047, an inhibitor of the prolyl-oligopeptidase (POP), known to modulate angiogenesis, in an in vivo U87-xenograft model and in an in vitro study on human GB cells. Our results showed that KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg was able to reduce tumor burden in the xenograft-model. Moreover, KYP-2047 significantly reduced vascular endothelial-growth-factor (VEGF), angiopoietins (Ang) and endothelial-nitric-oxide synthase (eNOS) expression. In vitro study revealed that KYP-2047 at different concentrations reduced GB cells' viability. Additionally, KYP-2047 at the concentrations of 50 µM and 100 µM was able to increase the pro-apoptotic protein Bax, p53 and caspase-3 expression whereas Bcl-2 expression was reduced. Thus, KYP-2047 could represent a potential therapeutic treatment to counteract or reduce GB progression, thanks its abilities to modulate angiogenesis and apoptosis pathways.
| S-EPMC8306782 | biostudies-literature
Project description:Cyclotides are head-to-tail cyclized peptides comprising a stabilizing cystine-knot motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 ?M. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 ?M. The inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson's disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability.
| S-EPMC4444998 | biostudies-literature
Project description:Accumulation of misfolded proteins in the brain is a common hallmark of most age-related neurodegenerative diseases. Previous studies from our group identified the presence of anti-inflammatory and antioxidant compounds in leaves derived from the Chilean berry Ugni molinae (murtilla), in addition to show a potent anti-aggregation activity in models of Alzheimer´s disease. However, possible beneficial effects of berry extracts of murtilla was not investigated. Here we evaluated the efficacy of fruit extracts from different genotypes of Chilean-native U. molinae on reducing protein aggregation using cellular models of Huntington´s disease and assess the correlation with their chemical composition. Berry extraction was performed by exhaustive maceration with increasing-polarity solvents. An unbiased automatic microscopy platform was used for cytotoxicity and protein aggregation studies in HEK293 cells using polyglutamine-EGFP fusion proteins, followed by secondary validation using biochemical assays. Phenolic-rich extracts from murtilla berries of the 19-1 genotype (ETE 19-1) significantly reduced polyglutamine peptide aggregation levels, correlating with the modulation in the expression levels of autophagy-related proteins. Using LC-MS and molecular network analysis we correlated the presence of flavonoids, phenolic acids, and ellagitannins with the protective effects of ETE 19-1 effects on protein aggregation. Overall, our results indicate the presence of bioactive components in ethanolic extracts from U. molinae berries that reduce the load of protein aggregates in living cells.
| S-EPMC8320977 | biostudies-literature
Project description:Prolyl oligopeptidase (POP) is a large 80 kDa protease, which cleaves oligopeptides at the C-terminal side of proline residues and constitutes an important pharmaceutical target. Despite the existence of several crystallographic structures, there is an open debate about migration (entrance and exit) pathways for ligands, and their coupling with protein dynamics. Recent studies have shown the capabilities of molecular dynamics and classical force fields in describing spontaneous binding events and nonbiased ligand migration pathways. Due to POP's size and to the buried nature of its active site, an exhaustive sampling by means of conventional long enough molecular dynamics trajectories is still a nearly impossible task. Such a level of sampling, however, is possible with the breakthrough protein energy landscape exploration technique. Here, we present an exhaustive sampling of POP with a known inhibitor, Z-pro-prolinal. In >3000 trajectories Z-pro-prolinal explores all the accessible surface area, showing multiple entrance events into the large internal cavity through the pore in the ?-propeller domain. Moreover, we modeled a natural substrate binding and product release by predicting the entrance of an undecapeptide substrate, followed by manual active site cleavage and nonbiased exit of one of the products (a dipeptide). The product exit shows preference from a flexible 18-amino acid residues loop, pointing to an overall mechanism where entrance and exit occur in different sites.
| S-EPMC4286615 | biostudies-literature
Project description:Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.
| S-EPMC5397884 | biostudies-literature
Project description:Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.
| S-EPMC8759985 | biostudies-literature
Project description:Prolyl oligopeptidase (PREP) accelerates the aggregation of ?-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.
| S-EPMC4335246 | biostudies-literature